CN109580952A - A kind of markers for breast cancer hMAM test strip and preparation method thereof - Google Patents
A kind of markers for breast cancer hMAM test strip and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to breast cancer diagnosis technical fields, and in particular to a kind of markers for breast cancer hMAM test strip and preparation method thereof.Detection of Tumor Markers in Patients with Breast Carcinoma hMAM test strip, including bottom plate, sample pad, microballoon pad, nitrocellulose filter and water absorption pad, successively overlapping overlap joint is arranged on the bottom plate from beginning to end for the sample pad, microballoon pad, nitrocellulose filter and water absorption pad, hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound is fixed on the microballoon pad, detection line T line and nature controlling line C line are coated on the nitrocellulose filter, it is coated with hMAM monoclonal antibody Ab1 at the T line, is coated with sheep anti-mouse antibody Ab2 at the C line.Detection of Tumor Markers in Patients with Breast Carcinoma hMAM test strip can realize the quick detection of hMAM in blood sample.
Description
Technical field
The present invention relates to breast cancer diagnosis technical fields, and in particular to a kind of markers for breast cancer hMAM test strip and
Preparation method.
Background technique
Breast cancer (breast cancer) is the complexity as caused by a variety of carcinogenic factors and different substantiality disease, accounts for women cancer
The 29% of sum occurs for disease, occupies the second (14%) of the female cancer death rate.Although the treatment method and effect of current breast cancer
Fruit has significant improvement and is promoted, but as the outcome of triple negative breast cancer etc. is still undesirable.Therefore, the early stage sieve of breast cancer
Looking into treatment is always the hot spot studied, but previously lacks specific Testing index and ideal detection method always.
For breast cancer, the index for clinically detecting breast cancer at present mainly has Cyfra21-1 (CK19), cancer embryo
Antigen (CEA), CA153, P53, BRCA-1 (breast cancer susceptibility gene -1), BRCA-2 (breast cancer susceptibility gene -2) etc., but this
A little tumor markers lack Breast Cancer-Specific and sensibility.Research shows that Human mammaglobin (human
Mammaglobin, hMAM) it is a kind of tumor markers of ideal diagnosis early-stage breast cancer, therefore establish and be based on hMAM
The breast cancer detection reagent of detection has great importance to the diagnosis, transfer, prognosis etc. of breast cancer.
Human mammaglobin belongs to uteroglobin (uteroglobin, UG) superfamily, is 93 Amino acid profiles
Polypeptide, molecular weight 10.5kDa.Most of researchs all confirm that hMAM gene is only expressed in adult mammary gland, swollen in many mammary gland
It is over-expressed in tumor, thus can be used as mammary gland and Breast Cancer-Specific marker.Since breast tissue blood supply is abundant, breast cancer cancer
Cellular infiltration blood vessel is simultaneously a kind of Main Patterns of Metastasis in Breast Cancer along bloody path transfer, or even can still deposit after ocal resection
It is in systemic circulation, and hMAM is a kind of potential secreted protein, can detecte depositing for it in blood serum of patients with human breast carcinoma
, this just make it possible based on breast cancer serum check.Therefore peripheral blood hMAM albumen rapid detection method is established, it can
For breast cancer relapse and the monitoring of transfer, it is particularly possible to which the generation for predicting small transfer instructs clinician to breast cancer disease
Early discovery, early diagnosis and the early treatment of disease.
It there is no effective hMAM rapid detection method at present.Metastasis of cancer is the first cause for causing patient with breast cancer's death.
Even if but high-resolution imaging technique can not detect the early stage transfer of tumour cell at present.Flow cytometry, RT-PCR,
The table of the cancer cell and oncogene that there is ELISA etc. the detection method compared with hypersensitivity can be used to spread in screening peripheral blood
It reaches.It is relatively common with screening breast cancer micrometastasis using hMAM mRNA expression in RT-PCR detection serum, but this method also has one
Fixed defect: mRNA is degradable, causes blood preparation that cannot save for a long time, and cannot obtain a large amount of cream in a short time
Adenocarcinoma patients' blood preparation is used for fairly large detection, statistics and analysis.ELISA method can be with quantitative detection, but each
It is required to establish standard curve when detection, is not suitable for the detection of breast cancer small sample.Therefore, there is an urgent need to establish a kind of suitable base
Medical institutions' hMAM rapid detection method.
POCT is the important development direction of Serologic detection, and POCT (Point Of Care Testing) is laboratory medicine
The frontier of development has the characteristics that fast and convenient, field assay as a kind of new clinical detection means, is able to comply with existing
The allegro working method of generation society simultaneously meets personalized service request.Time-resolved fluoroimmunoassay chromatography (TRFIA) is benefit
It is marked with the fluorescent microsphere facedown body containing lanthanide series, the dense of substance to be checked is analyzed by the power of fluorescence
Degree, can accurate quantitative analysis, the high sensitivity of detection, and can by easy fluorescence coating analyzer realize fast quantification, become blood
It is clear to learn one of detection important method, it is a kind of preferable in-vitro diagnosis method.
A kind of comprehensive detection box for breast cancer screening of CN 104931701A, although described in claim 1 by
Mammaglobin monoclonal antibody is coated in nitrocellulose filter as a wherein detection line, and using gold conjugation pad
Reaction card, but specific embodiment relevant to mammaglobin is not recorded in its specification.It has had not yet to see and has been used for
The rapid detection method of detection Human mammaglobin hMAM discloses.
Summary of the invention
The present invention provides a kind of markers for breast cancer hMAM test strip, to realize that markers for breast cancer hMAM's is quick
Detection.
The present invention also provides the preparation methods of above-mentioned markers for breast cancer hMAM test strip.
Markers for breast cancer hMAM test strip of the invention adopts the following technical scheme that a kind of markers for breast cancer
HMAM test strip, including bottom plate, sample pad, microballoon pad, nitrocellulose filter and water absorption pad, the sample pad, microballoon pad,
Successively overlap joint is arranged on the bottom plate from beginning to end for nitrocellulose filter and water absorption pad, is fixed with hMAM monoclonal on the microballoon pad
Antibody A b1 '-fluorescent microsphere compound is coated with detection line T line and nature controlling line C line, the T line on the nitrocellulose filter
Place is coated with hMAM monoclonal antibody Ab1, is coated with sheep anti-mouse antibody Ab2 at the C line.
Preferably, the lap-joint of the lap-joint of the sample pad and microballoon pad, the microballoon pad and nitrocellulose filter with
And the nitrocellulose filter and the be overlapped 1-2mm of the lap-joint of water absorption pad.
Preferably, hMAM monoclonal antibody the Ab1 '-fluorescent microsphere compound by fluorescent microsphere in buffer through EDC
It is coupled with activating solution and hMAM monoclonal antibody Ab1 ' concussion obtained after NHS activation, adds redissolution liquid to redissolve, then seal up and close fluid-tight
It closes and is made.
Preferably, the buffer is borate buffer.
Preferably, the redissolution liquid includes buffer, bio-activity protector, surfactant and microbial inhibitor,
Any one or a few in PVP, PEG-200, Triton X-100 or glycine of the surfactant, the life
Object activity protecting agent is selected from BSA and trehalose/sucrose, and the microbial inhibitor is NaN3.Dissolution Dan Ke can be played by redissolving liquid
The effect of grand antibody A b1 '-fluorescent microsphere compound, redissolution liquid of good performance can be in dissolution monoclonal antibody Ab1 '-
The line for being dissolved more evenly when fluorescent microsphere compound, and then detecting markers for breast cancer hMAM test strip
Property range is wider, and non-specific adsorption is less.Such as redissolution liquid can are as follows: redissolution formula of liquid 1:0.05M PH 8.0Tris-HCL,
0.1%
PVP (mass percent), 1%BSA (mass percent), 3% trehalose (mass percent), 0.5% glycine
(mass percent), 0.1%Tween-20 (mass percent), 0.05%PEG-200 (mass percent) and 0.05%
NaN3(mass percent, the concentration of above-mentioned each component are final concentration)；Redissolve formula of liquid 2:0.05M PH
8.0Tris-HCl, 0.1%PVP (mass percent), 0.5%BSA (mass percent), 3% trehalose (mass percent),
1%
Triton X-100 (mass percent) and 0.05%NaN3(concentration of mass percent, above-mentioned each component is
Final concentration) etc..
Preferably, the confining liquid is mass fraction 5%-15%BSA.
Preferably, the microballoon is lined with glass fibre membrane after treatment fluid is handled, then fixes on the glass fibre membrane
Monoclonal antibody Ab1 '-fluorescent microsphere compound is made, and the treatment fluid includes buffer, sucrose/trehalose, BSA and surface
Activating agent, any one or a few in PVP, PEG-200, Triton X-100 or glycine of the surfactant.
For example, matching for treatment fluid can are as follows: prescription for the treatment of liquid 1:0.1M PH 8.0Tris-Hcl, 0.1%Tween-20 (quality percentage
Number), 0.5%~1%BSA (mass percent), 3% trehalose (mass percent), 0.05%NaN3(mass percent, it is above-mentioned
The concentration of each component is final concentration)；Formula 2 for 0.05M PH 8.0Tris-HCL, 0.5%BSA (mass percent),
0.1%Tween-20 (mass percent), 3% sucrose (mass percent), 0.05%NaN3(mass percent, above-mentioned each group
The concentration divided is final concentration)；Treatment fluid, which has, promotes glass fibre membrane and monoclonal antibody Ab1 '-fluorescent microsphere compound knot
The effect of conjunction makes fluorescent microsphere being more evenly distributed on glass fibre membrane.It is demonstrated experimentally that (the 0.05M PH of prescription for the treatment of liquid 2
8.0Tris-HCL, 0.5%BSA, 0.1%Tween-20,3% sucrose, 0.05%NaN3) effect it is more preferable.
Preferably, the concentration of hMAM monoclonal antibody Ab1 is 1.0mg/mL-2.0mg/mL at the T line, at the C line
The concentration of sheep anti-mouse antibody Ab2 is 1.0mg/mL-2.0mg/mL.
A kind of preparation method of markers for breast cancer hMAM test strip as described above includes the following steps: (1) point
Microballoon pad and the nitrocellulose filter for being coated with T line and C line are not prepared；(2) viscous at the both ends of the nitrocellulose filter respectively
Microballoon pad and water absorption pad are pasted, the microballoon pad and water absorption pad are laminated on the top of nitrocellulose filter both ends end respectively；
(4) sample pad is laminated on to the top of the described one end of microballoon pad far from the nitrocellulose filter.
Preferably, the preparation of the microballoon pad includes the following steps: that (1) activates microballoon: it is micro- that fluorescence being added into buffer
Ball, ultrasound；EDC and NHS activator ultrasound is added, is vortexed and mixes；Oscillation activation；Centrifugation, removes supernatant, and it is multiple that buffer is added
It is molten, obtain fluorescent microsphere activating solution；(2) hMAM monoclonal antibody Ab1 ' and fluorescent microsphere activating solution are coupled:
HMAM monoclonal antibody Ab1 ' vortex is added into the fluorescent microsphere activating solution to mix, ultrasound；Oscillation coupling, from
The heart,
Supernatant is removed, is added into precipitating and redissolves liquid redissolution, ultrasound；Confining liquid is added, oscillation is vortexed and mixes, it is mono- to obtain hMAM
Clonal antibody Ab1 '-fluorescent microsphere compound；(3) hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound is fixed on described
On microballoon pad: it is glimmering that hMAM monoclonal antibody Ab1 '-being at the uniform velocity added dropwise on the glass fibre membrane obtained after treatment fluid is handled in advance
Light microsphere compound, it is dry to get microballoon pad.Ultrasonic treatment (in buffer be added microballoon after, be added activator EDC and NHS
Afterwards, hMAM monoclonal antibody Ab1 ' is added afterwards) combination of fluorescent microsphere and hMAM monoclonal antibody Ab1 ' can be made more evenly, in turn
So that the accuracy of obtained markers for breast cancer hMAM test strip is more preferable.
Markers for breast cancer hMAM test strip of the invention may also be fabricated which detection card or detection box: by breast cancer mark
In the setting of object hMAM test strip is got stuck in plastics, plastics get stuck counter sample pad position at well, corresponding nitre are set
Observation window is set at the position of acid cellulose film, preferably to protect markers for breast cancer hMAM test strip, reduce due to
Other substances in operating environment influence the pollution of test strips caused by testing result, are also convenient for hand-held test paper when detection
Item.
In addition, markers for breast cancer hMAM test strip of the invention can also be used for and disposable blood taking needle, disinfection dress
It is made into together with the instrument combination set, used in the detection process such as sample collection bottle, pipette and fluorescence immune chromatography readout instrument
Box is detected, so that more convenient when using.
The principle of markers for breast cancer hMAM test strip of the invention: according to the principle of double antibody sandwich method, when to
When containing hMAM in test sample sheet, hMAM-hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound (will be coated with by T line simultaneously
HMAM monoclonal antibody Ab1, monoclonal antibody Ab1 and monoclonal antibody Ab1 ' can be in conjunction with epitopes different from hMAM's) and C
Line (being coated with sheep anti-mouse antibody Ab2) capture generates the compound for having fluorescent marker, has at T line and C line under ultraviolet light irradiation
Fluorescent bands, testing result are the positive；Conversely, then only there are fluorescent bands in C line position when being free of hMAM in detection sample,
Testing result is feminine gender；Do not go out if fluorescence shows band if T line and C line and illustrates to detect invalid (referring to Figure of description Fig. 5).
The deeper hMAM content for illustrating to contain in sample to be tested of band color is higher at T line.
The beneficial effects of the present invention are: (1) markers for breast cancer hMAM test strip of the invention can fast quantification/fixed
Property detection human serum in hMAM content, detection time greatly shortens, and provides foundation for early diagnosing mammary cancer；Mammary gland of the invention
Carcinoma marker hMAM test strip can be applied to community's basic hospital without larger medical detection device, can promote cream
Gland cancer early screening rate, has a wide range of application.
(2) detection sensitivity of markers for breast cancer hMAM test strip of the invention is good, the range of linearity is wide and accurate
Property is good.The range of linearity is 6.25ng/ml-400ng/ml, cut off value are as follows: 8.63ng/ml.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram of markers for breast cancer hMAM test strip of the invention；
Fig. 2 is the breast cancer mark that the method according to the invention distinguishes that two kinds of redissolution formula of liquid (remaining processing is all the same) obtain
The testing result comparison diagram of will object hMAM test strip；
Fig. 3 is to be ultrasonically treated and do not surpass in preparation hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound preparation process
The testing result comparison diagram for the markers for breast cancer hMAM test strip that sonication (remaining processing is all the same) obtains；
Fig. 4 is the hMAM concentration-drawn using markers for breast cancer hMAM test strip prepared by method of the invention
T/C value canonical plotting；
Fig. 5 is the result judgement that sample detection after detection card is made in markers for breast cancer hMAM test strip of the invention
Figure；
Fig. 6 is the ELISA kit testing result linear comparison chart of the invention with existing detection hMAM on the market；
In figure: 1- sample pad；2- bonding pad；3- nitrocellulose filter；4-T line；5-C line；6- water absorption pad；7- bottom plate.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The preparation of the immuno-chromatographic test paper strip of 1 hMAM of embodiment
The preparation of 1.1 microballoon pads
1.1.1 hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound preparation
Take 8.0 borate buffer of 400ul 0.05M pH that 5ul microballoon, ultrasound 2 minutes is added.Add 10ul activator 1mg/
Ml EDC (ready-to-use), 1mg/ml NHS, ultrasound 1 minute, vortex mix, and are put into shaking table low-speed oscillation activation 30min；Activation
15000r/min is centrifuged 15min afterwards, abandons supernatant, and 500ul 0.05M pH8.0 borate buffer is added and redissolves precipitating, vortex is mixed
It is even, ultrasound 5 minutes.It is added 5ug hMAM monoclonal antibody (Ab1 '), is vortexed and mixes, ultrasound 1 minute.It is put into shaking table low-speed oscillation
It is coupled 2h.15000r/min is centrifuged 15min, abandons supernatant, 1250ul is added redissolves liquid and redissolve, then with 20ul confining liquid (10%
BSA 30min) is closed, the composition (redissolving formula of liquid 1) for redissolving liquid is 0.05M PH 8.0Tris-HCL, 0.1%PVP (matter
Measure score), 1%BSA (mass fraction), 3% trehalose (mass fraction), 0.5% glycine (mass fraction), 0.1%
Tween-20 (mass fraction), 0.05%PEG-200 (mass fraction) and 0.05%NaN3(mass fraction, above-mentioned each component
Concentration is final concentration).
1.1.2 hMAM monoclonal antibody Ab1 '-fluorescent microsphere is composite and fixed on glass fibre membrane
It is roomy small that glass fibre membrane is cut into 30mm*7mm, adds 64ul treatment fluid to carry out immersion treatment according to every 1cm, pulls out
Dry 2h is placed in electric drying oven with forced convection, it is using three-dimensional point film gold spraying instrument that the hMAM monoclonal antibody Ab1 '-of preparation is glimmering
Light microsphere compound adds the ratio uniform of 60ul to be sprayed on the glass fibre membrane handled well, is placed in drying room according to every 1cm,
In 37 DEG C of temperature, the lower dry 2h of humidity 10%, it is prepared into bonding pad；The group for the treatment of fluid become 0.05M PH 8.0Tris-HCL,
0.5%BSA (mass fraction), 0.1%Tween-20 (mass fraction), 3% sucrose (mass fraction, the concentration of above-mentioned each component
It is final concentration).
1.2 are coated with T line and C line on nitrocellulose filter
Using 0.05M PBS dilution hMAM monoclonal antibody (Ab1) and mark；Nitrocellulose filter is pasted onto bottom plate
The middle position of (PVC board), setting three-dimensional point film gold spraying instrument scribing line speed 10cm/s, cross concentration 1.0ul/cm.It is wrapped at T line
By 1mg/ml~2.0mg/ml hMAM monoclonal antibody (Ab1), 1mg/ml~2.0mg/ml sheep anti mouse secondary antibody is coated at C line
(Ab2), dry 2h is subsequently placed at 37 DEG C.
The assembling of 1.3 test strips:
Sample pad, microballoon pad, nitrocellulose filter and water absorption pad are successively overlapped and are assembled in bottom plate (PVC board), wherein
Water absorption pad and bonding pad overlap the both ends (being overlapped 1-2mm respectively) for being pressed in nitrocellulose filter respectively, and in nitrocellulose filter
Surface formed detection zone；Sample pad is overlapping to be pressed on bonding pad (overlapping 1-2mm), and the test paper of wide 3.9mm is cut into after assembling
Item is put into the aluminium foil bag added with desiccant.
0.39 the detection limit and the range of linearity of 1.4 test strips: a series of hMAM titer of concentration gradients is configured: 0,
0.78,1.56,3.125,6.25,12.5,25,50,100,150,200,300,400 (ng/ml) takes 70uL to be added drop-wise to immune layer
It analyses in test strips, reads the fluorescence intensity of T line and C line after 5min with detector, respectively by the fluorescence intensity of T/C and corresponding
HMAM concentration of standard solution does matched curve, obtains fluorescence intensity formula corresponding with concentration.Linear fit curve is shown in that specification is attached
The lines of triangle mark, calibration curve equation y=0.032x+0.25, R are used in Fig. 22=0.992.
Embodiment 2: liquid replacement will be redissolved are as follows: 0.05M PH 8.0Tris-HCl, 0.1%PVP, 0.5%BSA, 3% seaweed
Sugar, 1%Triton X-100,0.05%NaN3(redissolve formula of liquid 2), remaining operation and used reagent with embodiment one
It is identical, to the hMAM titer of following concentration gradients: 0,0.39,0.78,1.56,3.125,6.25,12.5,25,50,100,
150,200,300,400 (ng/ml) are detected (detection method is with embodiment one), respectively that the fluorescence intensity of T/C is (vertical to sit
Mark) and corresponding hMAM concentration of standard solution (abscissa) do matched curve, obtain fluorescence intensity formula corresponding with concentration.Standard
Curvilinear equation is y=0.026x+0.2319, R2=0.9793, linear fit curve is shown in Figure of description 2 with dot label
(redissolve liquid is that obtained calibration curve equation and linear fit curve are shown in Figure of description Fig. 2 when redissolving formula of liquid 1 to lines
With the lines of triangle mark.Redissolution formula of liquid 1 is 0.05M PH 8.0Tris-HCL, 0.1%PVP, 1%BSA, 3% seaweed
Sugar, 0.5% glycine, 0.1%Tween-20,0.05%PEG-200,0.05%NaN3)。
Conclusion: redissolve liquid group be divided into redissolve formula of liquid 1 (0.05M PH 8.0Tris-HCL, 0.1%PVP, 1%BSA,
3% trehalose, 0.5% glycine, 0.1%Tween-20,0.05%PEG-200,0.05%NaN3) when, hMAM monoclonal is anti-
More evenly, the detection range of linearity of obtained test strips is wider for the dissolution of body Ab1 '-fluorescent microsphere, non-specific adsorption compared with
It is few.It is detailed in Figure of description Fig. 2.
Embodiment 3: hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound system is detected respectively with the standard items of 50ng/ml
Test strips (other preparation methods and agents useful for same of test strips obtained under ultrasonic and not ultrasonic two kinds of dispositions during standby
It is same as Example 1), it detects 10 times respectively.By CV=SD/X, if obtaining, hMAM monoclonal antibody Ab1 '-fluorescent microsphere is multiple
It closes without ultrasound in object preparation process, testing 10 obtained CV values is 7.2%；If hMAM monoclonal antibody Ab1 '-fluorescence
Be ultrasonically treated in microsphere compound preparation process (in buffer be added microballoon after, be added activator EDC and NHS after, plus
Enter hMAM monoclonal antibody Ab1 ' afterwards), testing 10 obtained CV values is 3.2%, and specific experiment data are detailed in Figure of description
Fig. 3.It follows that ultrasonic treatment can make the combination of fluorescent microsphere and hMAM monoclonal antibody Ab1 ' more evenly, the essence of test strips
Close property is more preferable.
Embodiment 4: test strips performance verification of the invention:
4.1 ranges of linearity:
The test strips being prepared using embodiment 1 respectively to the linear quality-control product of each concentration (0,0.39,0.78,1.56,
3.125,6.25,12.5,25,50,100,150,200,300,400ng/mL) detection 10 times is repeated, calibration curve is drawn, through number
According to fitting and statistical analysis, calibration curve equation y=0.0339x+0.2246, R are obtained2=0.9962 (is detailed in Figure of description figure
4), test paper linear detection range of the present invention is 6.25ng/ml-400ng/ml.Above-mentioned standard curve is set in advance in fluorescence to exempt from
Epidemic disease chromatographs in readout instrument, when quantitative detection, takes 65 μ L of blood serum sample to be added in well, test strips are put into fluorescence immune chromatography
In readout instrument, test strips read fluorescence intensity level in fluorescent chromatographic readout instrument after 5min, (draw out standard song according to standard curve
Can be pre-stored in fluorescence immune chromatography readout instrument after line, can directly read the content of hMAM in institute's test sample) it calculates
HMAM content.
4.2 qualitative detections: the cut off value of known enzyme-linked immunosorbent assay (ELISA) detection breast cancer serum hMAM
Are as follows: 8.63ng/ml, if the concentration of hMAM is greater than 8.63ng/mL, when using test strips qualitative detection of the invention, T line in sample
With have fluorescence at C line, therefore test strips prepared by the present invention can satisfy the inspection of normal person and blood serum of patients with human breast carcinoma hMAM
It surveys.It takes 65 μ L of blood serum sample to be added in well when detection, test strips is placed under ultraviolet lamp after 5min, interpretation result.According to
The principle of double antibody sandwich method, when containing hMAM in sample to be tested, hMAM-hMAM monoclonal antibody Ab1 '-fluorescent microsphere is multiple
Closing object will be captured by T line and C line simultaneously, generate the compound for having fluorescent marker, have at T line and C line under ultraviolet light irradiation glimmering
Striation band, testing result are the positive；Conversely, then only there are fluorescent bands in C line position when being free of hMAM in detection sample, examine
Surveying result is feminine gender；Do not go out if fluorescence shows band if T line and C line and illustrates to detect invalid (referring to Figure of description Fig. 5).T
The deeper hMAM content for illustrating to contain in sample to be tested of band color is higher at line.
4.2 accuracies:
Detect 3 kinds of accuracy quality-control products respectively using hMAM fluorescence immune chromatography test paper bar prepared by embodiment 1: hMAM is low
It is worth (5ng/ml), intermediate value (10ng/ml) and high level (50ng/ml), repeats detection 10 times respectively, carries out accuracy measurement.Utilize T
The coefficient of variation of line fluorescence intensityHigh level CV=5.1%, intermediate value CV=7.3%, low value CV=is calculated
8.8%, as a result shown in the following table 1:
Table 1
| Accuracy | Mean | Standard deviation | CV |
| 50ng/mL | 2.082020795 | 0.106564228 | 0.051183076 |
| 10ng/mL | 0.581519233 | 0.04261381 | 0.073280138 |
| 5ng/mL | 0.404216816 | 0.035750341 | 0.088443479 |
Test strips testing result CV value prepared by the present invention is respectively less than 15%, illustrates that the test strips accuracy is good.
Embodiment 5: methodology compares
Take 20 parts of blood through the various concentration that existing Human mammaglobin (hMAM) ELISA kit detected on the market
Clear sample is detected with test strips prepared by embodiment 1, is vertical with the hMAM concentration that the homemade test strips of embodiment 1 detect
Coordinate, the hMAM concentration detected using ELISA kit establishes dependent linearity analysis as abscissa, as shown in fig. 6, fitting obtains
The equation obtained is y=0.9367x+7.7372, coefficient R2It is 0.989, the two correlation is good.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of markers for breast cancer hMAM test strip, including bottom plate, sample pad, microballoon pad, nitrocellulose filter and suction
Water cushion, successively overlapping overlap joint is arranged on the bottom plate from beginning to end for the sample pad, microballoon pad, nitrocellulose filter and water absorption pad,
It is characterized by: being fixed with hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound, the nitrocellulose on the microballoon pad
It is coated with detection line T line and nature controlling line C line on film, hMAM monoclonal antibody Ab1 is coated at the T line, is coated at the C line
There is sheep anti-mouse antibody Ab2.
2. markers for breast cancer hMAM test strip according to claim 1, which is characterized in that the sample pad with it is micro-
The lap-joint of ball pad, the lap-joint of the microballoon pad and nitrocellulose filter and the nitrocellulose filter and water absorption pad are taken
It meets place and is overlapped 1-2mm.
3. markers for breast cancer hMAM test strip according to claim 1, which is characterized in that the hMAM monoclonal
Antibody A b1 '-fluorescent microsphere compound activating solution and hMAM obtained after EDC and NHS activation in buffer by fluorescent microsphere
Monoclonal antibody Ab1 ' concussion coupling adds and redissolves liquid and redissolve, then seals up to close fluid-tight and close and be made.
4. markers for breast cancer hMAM test strip according to claim 3, which is characterized in that the buffer is boron
Acid buffer.
5. markers for breast cancer hMAM test strip according to claim 3, which is characterized in that the redissolution liquid includes
Buffer, bio-activity protector, surfactant and microbial inhibitor, the surfactant be selected from PVP, PEG-200,
Any one or a few in Triton X-100 or glycine, the bio-activity protector is selected from BSA and trehalose/sugarcane
Sugar, the microbial inhibitor are NaN3。
6. markers for breast cancer hMAM test strip according to claim 3, which is characterized in that the confining liquid is matter
Measure score 5%-15%BSA.
7. markers for breast cancer hMAM test strip according to claim 1, which is characterized in that the microballoon pad is by glass
First immobilized monoclonal antibody Ab1 '-fluorescent microsphere is compound after treatment fluid is handled, then on the glass fibre membrane for glass tunica fibrosa
Object is made, and the treatment fluid includes buffer, sucrose/trehalose, BSA and surfactant, and the surfactant is selected from
Any one or a few in PVP, PEG-200, Triton X-100 or glycine.
8. markers for breast cancer hMAM test strip according to claim 1, which is characterized in that hMAM at the T line
The concentration of monoclonal antibody Ab1 is 1.0mg/mL-2.0mg/mL, and the concentration of sheep anti-mouse antibody Ab2 is 1.0mg/ at the C line
mL-2.0mg/mL。
9. the preparation method of markers for breast cancer hMAM test strip described in any one of -8 according to claim 1,
It is characterized in that, includes the following steps: that (1) prepares microballoon pad and the nitrocellulose filter for being coated with T line and C line, the nitre respectively
Acid cellulose film is pasted on bottom plate；(2) microballoon pad and water absorption pad are pasted at the both ends of the nitrocellulose filter respectively, it is described
Microballoon pad and water absorption pad are laminated on the top of nitrocellulose filter both ends end respectively；(4) sample pad is laminated on described
The top of the one end of microballoon pad far from the nitrocellulose filter.
10. the preparation method of markers for breast cancer hMAM test strip according to claim 9, which is characterized in that institute
The preparation for stating microballoon pad includes the following steps: that (1) activates microballoon: fluorescent microsphere being added into buffer, ultrasound；Be added EDC and
NHS activator, ultrasound are vortexed and mix；Oscillation activation；Centrifugation, removes supernatant, and buffer redissolution is added, and it is living to obtain fluorescent microsphere
Change liquid；(2) hMAM monoclonal antibody Ab1 ' and fluorescent microsphere activating solution are coupled: are added in Xiang Suoshu fluorescent microsphere activating solution
HMAM monoclonal antibody Ab1 ', which is vortexed, to be mixed, ultrasound；Supernatant is removed in oscillation coupling, centrifugation, and redissolution liquid is added into precipitating and redissolves,
Ultrasound；Confining liquid is added, oscillation is vortexed and mixes, obtains hMAM monoclonal antibody Ab1 '-fluorescent microsphere compound；(3) hMAM is mono-
Clonal antibody Ab1 '-fluorescent microsphere compound is fixed on the microballoon pad: to the glass obtained after treatment fluid is handled in advance
HMAM monoclonal antibody Ab1 '-fluorescent microsphere compound is at the uniform velocity added dropwise on tunica fibrosa, it is dry to get microballoon pad.
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| CN111579768A (en) * | 2019-06-25 | 2020-08-25 | 山西康健恩生物科技有限公司 | Anti-mullerian hormone detection kit and preparation method thereof |
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