CN107589264A - Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof - Google Patents
Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof Download PDFInfo
- Publication number
- CN107589264A CN107589264A CN201710755819.2A CN201710755819A CN107589264A CN 107589264 A CN107589264 A CN 107589264A CN 201710755819 A CN201710755819 A CN 201710755819A CN 107589264 A CN107589264 A CN 107589264A
- Authority
- CN
- China
- Prior art keywords
- fluorescent dye
- her2
- growth factor
- epidermal growth
- human epidermal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of (HER2) two-photon fluorescence immune chromatography reagent kit of quantitative inspection human epidermal growth factor receptor 2 using fluorescent dye as label.The fluorescence immune chromatography kit of the present invention realizes quantitatively to be detected to the two-photon fluorescence of human epidermal growth factor receptor 2 (HER2), with stability is good, the range of linearity is wide, specificity is good, high sensitivity, quantitative accurate and simple and quick advantage, whole blood, serum and plasma sample can be detected simultaneously, and are applied to situation of all-level hospitals and domestic medicine.
Description
Technical field
The present invention relates to the cancer clinical immunodiagnosis kit field in field of medical examination, and in particular to quantitative detection
The two-photon fluorescence immune chromatography reagent kit of human epidermal growth factor receptor 2 (HER2).
Background technology
Human epidermal growth factor receptor 2 (HER2/neu) is one of tyrosine kinase receptors family member, mainly by Ras/MAPK,
The paths such as PI3K/Akt participate in the propagation and anti-apoptotic of cell, by promoting matrix metalloproteinase (MMPs) to secrete, activate
VEGF promoter, while serine/threonine kinase (AKT) and NF-κappa B (NF-KB) etc. are activated, promote the leaching of tumour
Profit and transfer.The overexpression of HER2/neu albumen and gene magnification are present in kinds of tumors, including breast cancer, lung cancer, ovary
Cancer and tumor in digestive tract etc..It is related to generation, development, transfer, treatment and the prognosis of tumour.
1.HER2/neu and breast cancer
Research finds that 20%-30% patient with breast cancer has HER2/neu overexpression, and HER2/neu overexpression is normal
Prompt the grade malignancy of tumour high.Result of study show HER2/neu genes amplification can cause tumor recurrence and clinical prognosis compared with
Difference, and point out that the judgement to prognosis such as this hormone receptor, tumor size is more meaningful.HER2 expression and the pathology class of breast cancer
Type is proportionate negatively correlated with differentiation degree.HER2/neu expression is used as breast cancer independent prognostic index and selection
The major criterion for the treatment of.
2.HER2/neu and oophoroma
Oophoroma has the overexpression about 15%-30%'s of HER2/neu albumen.In normal ovarian and borderline ovary
Can't detect HER2/neu amplifications and overexpression in tumour, and in oophoroma common HER2/neu amplification and albumen it is excessive
Expression.Verri results of study show that HER2/neu protein overexpressions add ovarian cancer patients tumour progression and dead danger.
Many researchs show at present, and HER2/neu is overexpressed to be reduced with advanced ovarian cancer overall survival and recurrence time shortening is relevant, is
One of prognostic factor for the treatment of of ovarian cancer result badly.
3.HER2/neu and lung cancer
There is certain correlation with the type of lung cancer for HER2/neu mutation.Research thinks that HER2/neu mutation are mainly deposited
In bronchioloalveolar gland cancer.Also the research of someone shows that overexpression rates of the HER2/neu in cancerous lung tissue is
26.67%.Wherein mainly it is expressed in adenocarcinoma of lung, expression is showed no in ED-SCLC and squamous carcinoma.Its research also passes through observation
Influence of the cancer therapy drug to the lung adenocarcinoma cell multiplication capacity of different HER2/neu expressions, show the height of HER2/neu genes
One of the reason for expression is probably chemotherapy resistance, and prompt HER2/neu overexpression uncorrelated to lung cancer differentiation degree.And
Tumor center of university pharmacology is found in Colorado, Pathological Staff Room research shows, expression rates of the HER2/neu in lung cancer
For 7%-22.8%, cell differentiation is poorer, then positive rate is higher.
In summary, there are many tumours including breast cancer in the amplification of the overexpression of HER2/neu albumen and gene
In, in close relations with tumour generation, transfer, treatment etc., therefore, blood HER2/neu detection can be to the grade malignancy of tumour
And therapeutic effect is evaluated.
At present, the HER2 detections immunization method clinically commonly used has ELISA, chemoluminescence method, electrochemical luminescence
Method etc.；Because ELISA needs enzyme to mark, process is complicated, it is necessary to the human users of specialty, and U.S. linked immunosorbent assay position liquid
Body reagent brings very big inconvenience for basic medical unit, it is necessary to deepfreeze transport；Tube-type chemical luminescence method and electrochemistry
Luminescence method is to need large scale equipment, and is closed system, expensive, is unfavorable for the diagnosis and treatment of basic medical unit.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned the deficiencies in the prior art, there is provided a kind of basic hospital to Grade A hospital
Being applicable, high sensitivity, accuracy diagnostic reagent that is strong, easy to operate, being capable of quick diagnosis breast cancer, lung cancer and oophoroma
Box.
In order to solve the above technical problems, the present invention uses fluorescence immune chromatography technology, with human epidermal growth factor receptor 2
(HER2) Testing index is used as, specific technical scheme is：
One aspect of the present invention provides a kind of fluorescence immunoassay layer for quantitatively detecting human epidermal growth factor receptor 2 (HER2)
Kit, including buckle (11), two-photon fluorescence immuno-chromatographic test paper strip and buffer solution are analysed, wherein, buckle (11) is exempted from for fluorescence
The outside shell structure of epidemic disease chromatograph test strip, including well (9) and observation window (10), as shown in Figure 3.
Fluorescence immune chromatography test paper bar structure as depicted in figs. 1 and 2, including sample pad (1), label pad (2), chromatographic film
(6), adsorptive pads (7) and bottom plate (8).When carrying out whole blood sample detection, test strips also include blood separation membrane (3).Wherein, sample
Product pad (1), label pad (2), blood separation membrane (3), chromatographic film (6) and adsorptive pads (7) are equipped on bottom plate (8), sample pad
(1) it is located at the lower section of well (9), and is connected to label pad (2), label pad (2) is connected to chromatographic film (6), and chromatographic film (6) is even
Adsorptive pads (7) are connected to, are fixed with a quantitative detection line (4) and a nature controlling line (5) thereon, preferably the two distance 3mm~
8mm, and positioned at the lower section of observation window (10).In the case of including blood separation membrane (3), blood separation membrane (3) is arranged on mark
Between note pad (2) and chromatographic film (6), now label pad (2) is connected to blood separation membrane (3), and blood separation membrane (3) is connected to layer
Film (6) is analysed, or blood separation membrane (3) is arranged between sample pad (1) and label pad (2), now sample pad (1) is connected to blood
Liquid seperation film (3), blood separation membrane (3) are connected to label pad (2), or blood separation membrane (3) directly merges with sample pad (1)
For same structure.
Human epidermal growth factor receptor 2 (HER2) specific antibody of fluorescent dye modification is fixed with label pad (2) simultaneously,
With the Quality Control molecule of fluorescent dye modification, the hair of the fluorescent dye of (HER2) specific antibody of modification human epidermal growth factor acceptor 2
The launch wavelength that ejected wave grows the fluorescent dye with modifying Quality Control molecule is identical, wave-length coverage 300-500nm.
Human epidermal growth factor receptor 2 (HER2) specific antibody of fluorescent dye modification is fixed with quantitative detection line (4),
The antigenic determinant and the human epidermal growth factor for the fluorescent dye modification being fixed in label pad (2) that the specific antibody is identified
The antigenic determinant that acceptor 2 (HER2) specific antibody is identified is different.
The fluorescent dye modified biological molecule that can be combined with Quality Control molecular specificity or anti-mouse are fixed with nature controlling line (5)
Antibody.
Modify launch wavelength and the modification Quality Control point of the fluorescent dye of (HER2) specific antibody of human epidermal growth factor acceptor 2
The launch wavelength of the fluorescent dye of son is identical, wave-length coverage 500-800nm.
It is glimmering that another aspect of the present invention provides the above-mentioned two-photon for quantitatively detecting human epidermal growth factor receptor 2 (HER2)
The preparation method of light immune chromatography reagent kit, comprises the following steps：
Step (1)：Conjugated fluorescent dyes to people's epidermis are given birth to respectively with specific effect between chemical crosslinking or biomolecule
The surface of long (HER2) specific antibody of factor receptor 2 and Quality Control molecule, respectively obtain the human epidermal growth factor of fluorescent dye modification
(HER2) specific antibody of acceptor 2 and the Quality Control molecule of fluorescent dye modification, modify the hair of the fluorescent dye of the Quality Control molecule
The launch wavelength that ejected wave grows the fluorescent dye with modifying human epidermal growth factor acceptor 2 (HER2) specific antibody is identical, wavelength model
Enclose for 300~500nm；
Step (2)：Human epidermal growth factor receptor 2 (HER2) specificity that the fluorescent dye that step (1) is obtained is modified is anti-
Body and the Quality Control molecule of fluorescent dye modification are fixed in label pad (2)；
Step (3)：One quantitative detection line (4) and a nature controlling line (5) are set respectively in chromatographic film (6), wherein, matter
The biomolecule for the fluorescent dye modification that can be combined with Quality Control molecular specificity is fixed with control line (5), is quantified in detection line (4)
Human epidermal growth factor receptor 2 (HER2) specific antibody of fluorescent dye modification is fixed with, and the specific antibody is identified
Human epidermal growth factor receptor 2 (HER2) specificity of antigenic determinant and the fluorescent dye modification being fixed in label pad (2) is anti-
The antigenic determinant that body is identified is different；
Step (4)：Sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and bottom plate (8) are built into fluorescence
Immuno-chromatographic test paper strip, when carrying out whole blood sample detection, test strips also include blood separation membrane (3)；
Step (5)：Two-photon fluorescence immuno-chromatographic test paper strip is loaded.
The present invention is using specific effect between chemical crosslinking or biomolecule by conjugated fluorescent dyes to human epidermal growth factor
The surface of acceptor 2 (HER2) specific antibody or the surface of Quality Control molecule, respectively obtain the human epidermal growth of fluorescent dye modification
(HER2) specific antibody of factor receptor 2 and the Quality Control molecule of fluorescent dye modification.In the present invention, when fluorescent dye surface is present
During active group, it can directly be reacted with specific antibody, be not required to use chemical cross-linking agent；Conversely, then need to pass through chemical crosslinking
Fluorescent dye and (HER2) specific antibody of human epidermal growth factor receptor 2 or Quality Control molecule are mutually coupled by agent.Wherein, it is chemically crosslinked
Agent includes 1- ethyls -3- (3- DimethylAminopropyls) carbodiimides (EDC), n-hydroxysuccinimide (NHS) and glutaraldehyde
Deng.
In a preferred embodiment of the invention, using active group fluorescent dye modify human epidermal growth factor by
(HER2) specific antibody of body 2 or Quality Control molecule, step are：Fluorescent dye after purification is dissolved, then added a certain amount of
(HER2) specific antibody of human epidermal growth factor receptor 2 or Quality Control molecule, using buffer solution as reaction medium, reaction 2~4 is small
When, hydroxylamine hydrochloride terminating reaction is added, is purified with modes such as chromatogram, chromatographic column or ultrafiltration centrifugations, obtains fluorescent dye modification
(HER2) specific antibody of human epidermal growth factor receptor 2 or Quality Control molecule.
In order to improve the discrimination of signal and background, marked in the present invention wave-length coverage of fluorescent dye for 300~
500nm, this example select 365nm.Because under ultraviolet irradiation, the fluorescence intensity of chromatographic film, bottom plate and buckle 550nm with
Under can be much stronger than more than 550nm, so as to which certain shadow can be produced to the detection of low concentration human epidermal growth factor receptor 2 (HER2)
Ring, therefore preferred emission wavelength is more than 550nm fluorescent dye；In addition, chromatographic film, bottom plate and buckle are typically near infrared region
(600~800nm) fluorescence intensity is extremely weak, therefore, the fluorescent dye that coating fluorescent dye preferred wavelength range is 500-800nm with
Sensitivity is further improved, it is 635nm that this example, which selects fluorescent dye launch wavelength,.
The fluorescent dye of the present invention includes organic fluorescent dye and rare earth element fluorescent dye.The fluorescent dye of the present invention can
It is the form that becomes fluorescent microsphere on latex microsphere to be coupled to.The fluorescent dye of the present invention can be single compound
Fluorescent dye or by several compound groups into composite fluorescent dye, preferably single compound fluorescent dye and preferably have
The fluorescent dye of stronger photostability.The present invention fluorescent dye such as AlexaFluro series (AlexaFluro647,
610th, 633,700, fluorescein 750), DyLight serial (DyLight649,633,549,680) etc..
Human epidermal growth factor receptor 2 (HER2) specific antibody of the present invention is monoclonal antibody or polyclonal antibody.Matter
Control molecule such as rabbit igg, the biomolecule such as goat anti-rabbit igg combined with Quality Control molecular specificity.
In order to reduce the influence to fluorescent dye fluorescence signal, the present invention uses chromatographic film, bottom plate and the button of hypofluorescence
Card, its fluorescent noise be more than 550nm can be very weak, can good discrimination signal and the back of the body so as to ensure to obtain high fluorescence signal-to-background ratio
Scape, and then improve detection sensitivity.It is preferred that bottom plate is white, surface has an adhesive sticker, and buckle, chromatographic film, bottom plate and does not do
Glue does not contain fluorescer.
In the present invention, testing sample can be serum or blood plasma, and now fluorescence immune chromatography test paper bar is not including blood point
From film.Testing sample can also be whole blood, and now fluorescence immune chromatography test paper bar also includes blood separation membrane, for solidifying, mistake
Filter cell.Blood separation membrane may be provided between label pad and chromatographic film, respectively with label pad and the direct capillarity of chromatographic film
Contact, or be arranged between sample pad and label pad, also can be straight respectively with sample pad and the direct capillary contact of label pad
Connect and merge into same structure with sample pad, so as to which sample pad has the function that sample collection, release and filtering simultaneously.
The fluorescence immune chromatography kit of the present invention is determined the human epidermal growth factor receptor 2 (HER2) in testing sample
Amount detection.During detection, testing sample is added in sample pad by well, sample chromatographs along chromatographic film to adsorptive pads direction
Motion.The sample chromatography time is usually 8~25 minutes, preferably 15 minutes.After chromatography, with Wash buffer solution for cleaning chromatographic films,
Time is 3-8 minutes, preferably 5 minutes, to reduce background, improves detection sensitivity.Ox is included in the Wash buffer solutions of the present invention
Seralbumin, sucrose and surfactant, pH value 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is 0.05~2%,
The concentration of sucrose is 1~15%, and the concentration of surfactant is 0.05~2%.The preferred polysorbas20 of surfactant, Qula lead to X-
100, the preferred Tris-HCl buffer solutions of buffer solution, phosphate buffer.
The detection of the present invention includes excitation source module, filtration module, photoelectric conversion module, control point with fluorescent quantitation instrument
Analyse module and software systems.Wherein excitation source module includes light source and beam condensing unit, and the light source is light emitting diode or swashed
Optical diode, and wavelength is between 600~750nm.Filtration module includes optical filter wheel, and the optical filter wheel includes filter not of the same race
Mating plate, to obtain corresponding fluorescence signal.Photoelectric conversion module includes imaging sensor or photomultiplier.
After chromatography terminates, under light source activation, fluorescence signal caused by test strips filters out veiling glare and the back of the body through filtration module
Scape fluorescence, photoelectric conversion module is reached, obtain data signal, the fluorescence signal intensity that nature controlling line obtains obtains glimmering with detection line
Light signal strength has correlation：If nature controlling line fluorescence signal intensity exceeds the acceptable value of fluorescent quantitation instrument inner setting,
Illustrate that testing result is invalid；Under the premise of testing result is effective, fluorescence signal intensity and nature controlling line fluorescence that detection line obtains are believed
The ratio of number intensity is higher, represents that the concentration of target detection thing in sample is higher, otherwise lower.
The present invention detects a series of standard items of various concentrations using fluorescent quantitation instrument, draws standard curve.Its Plays
Curve is the relation curve of standard items series concentration (X) and corresponding fluorescence signal intensity (Y), relational expression Y=y0+bX,
Fluorescence signal intensity is Y=detection lines peak area/nature controlling line peak area, then detects sample, and establishing criteria curve obtains to be measured
The concentration of human epidermal growth factor receptor 2 (HER2) in sample.
The operation principle of fluorescence immune chromatography kit of the present invention is：Pressed from both sides using fluorescence immune chromatography technology and double antibody
Heart method principle quantitatively detects the content of the human epidermal growth factor receptor 2 (HER2) in sample (whole blood, serum or blood plasma).Detection
When, sample is added drop-wise in loading hole, is combined with fluorochrome label thing during the labeled pad of sample flow, and along chromatographic film to
Adsorptive pads direction capillary moving, flow separately through quantitative band and quality control band in chromatographic film.If contain human epidermal growth factor in sample
Acceptor 2 (HER2), then it is combined with human epidermal growth factor receptor 2 (HER2) antibody modified by fluorescent dye, when chromatography to inspection
During survey line, the capture antibody that can be coated in the band in advance captures, so as to form double-antibody sandwich compound.Light source activation
Under, the fluorescence signal intensity of detection line and nature controlling line can be obtained using fluorescent quantitation instrument, the standard obtained according to fluorescent quantitation instrument
Curve, and then the concentration containing HER2 in sample can be analyzed.
Main advantages of the present invention are as follows：
1) present invention adopts double organic fluorescent dye, rare earth element fluorescent dye or quantum dot fluorescence dyestuffs as specificity
The fluorescent marker of antibody, wherein short wavelength's fluorescent dye are launched photoemissive two photons and produced by long wavelength's fluorescent dye absorption
A raw long wavelength emission light, has that luminous intensity is high, emission spectrum is narrow, fluorescence lifetime is long, surface modification multifunction, stably
Property the advantage such as good and sensitivity height.
2) buckle, bottom plate and the chromatographic film in kit forms part of the present invention are respectively provided with low when more than 550nm
Fluorescent characteristic, it is possible to reduce the influence obtained to fluorescent dye fluorescence signal, so as to ensure to obtain high fluorescence signal-to-background ratio, and then
Reach and put forward highly sensitive purpose.
3) the two-photon fluorescence immune chromatography method of the present invention for quantitatively detecting human epidermal growth factor receptor 2 (HER2)
For Wash chromatographic techniques, non-specific adsorption can be reduced, reduces autofluorescent background, enhancing specific binding, and then improve detection spirit
Sensitivity, be advantageous to accurate quantitative analysis when human epidermal growth factor receptor 2 (HER2) content is extremely low in sample.
4) the inventive method is compared with conventional fluorescent immunochromatography, has that mark stability is good, non-specific low, sensitivity
Height, the range of linearity are wide and quantify the advantage such as accurate.
Brief description of the drawings
Fig. 1 is the assembling schematic diagram of fluorescence immune chromatography test paper bar, wherein 1 is sample pad, 2 be pad, and 3 be blood point
From film, 4 be detection line, and 5 be nature controlling line, and 6 be chromatographic film, and 7 be adsorptive pads, and 8 be bottom plate.
Fig. 2 is fluorescence immune chromatography test paper bar sample test schematic diagram in embodiment 1, and utilizes the kit test specimens
The detection peak and Quality Control peak that product obtain.
Fig. 3 is fluorescence immune chromatography kit schematic diagram, wherein 11 be buckle, 9 be well, and 10 be observation window, and 4 be inspection
Survey line, 5 be nature controlling line.
Fig. 4 is the standard curve of fluorescence immune chromatography kit in embodiment 1, with human epidermal growth factor receptor 2 (HER2)
Concentration is abscissa, with fluorescence intensity (detection peak area ratio Quality Control peak area ratio) for ordinate.
Embodiment
Embodiment 1：With fluorescent dye modified antibodies in a manner of covalent cross-linking, and Wash immunochromatography techniques are adopted to people's epidermis
Grow the quantitative detection of factor receptor 2 (HER2)
1) coupling of fluorescent dye and antibody
Human epidermal growth factor receptor 2 (HER2) monoclonal of fluorescent dye and l mg/ml that launch wavelength is 365nm is resisted
Body mixes, and reacts at room temperature 2-4h, adds 1mol/L hydroxylamine hydrochloride terminating reactions, and with chromatographic column or chromatographs column separating purification, obtains
To human epidermal growth factor receptor 2 (HER2) monoclonal antibody of fluorescent dye modification.Similarly obtain the rabbit igg of fluorescent dye modification
(Quality Control molecule).Wherein modify fluorescence emission wavelengths and the modification of the fluorescent dye of (HER2) antibody of human epidermal growth factor acceptor 2
The fluorescence emission wavelengths of the fluorescent dye of rabbit igg are 365nm.
By the fluorescent dye and l mg/ml coating human epidermal growth factor receptor 2 (HER2) Dan Ke that launch wavelength is 680nm
Grand antibody mixing, reacts at room temperature 3h, adds 1mol/L hydroxylamine hydrochloride terminating reactions, and with chromatographic column or chromatographs column separating purification,
Obtain human epidermal growth factor receptor 2 (HER2) monoclonal antibody of fluorescent dye modification.Similarly obtain supporting for fluorescent dye modification
Anti-rabbit IgG (Quality Control molecule).Wherein modify the fluorescence emission wavelengths of the fluorescent dye of (HER2) antibody of human epidermal growth factor acceptor 2
The fluorescence emission wavelengths of fluorescent dye with modifying rabbit igg are 680nm.
2) structure of kit
With 1:1 ratio mixes two kinds of fluorochrome label things, and adds bovine serum albumin(BSA) (content 1%), sucrose
(content 10%) and surfactant triton x-100 (content 0.8%), subsequent even application is in label pad, and 45
DEG C dry after seal, preserve at room temperature.
As shown in figure 1, assembling quantitatively detect HER2 fluorescence immune chromatography test paper bar, by sample pad (1), label pad (2),
Blood separation membrane (3), chromatographic film (6), adsorptive pads (7) composition, are sequentially pasted on white bottom plate (8).Wherein, sample pad is hole
Shape barrier film, glass fibre is selected, is testing sample collecting region；HER2 specific antibodies containing fluorescent dye modification in pad
And the rabbit igg of fluorescent dye modification；It is fixed with quantitative detection line (4) and nature controlling line (5) in chromatographic film, detection line and nature controlling line
At intervals of 5mm, and detection line (4) is fixed with the specificity for being different from another epitope of HER2 specific antibodies in label pad
Antibody, nature controlling line (5) are fixed with goat anti-rabbit antibody.After assembling, required width is cut into as requested, is placed in buckle (11)
It is interior, as shown in Fig. 2 adding drier encapsulation, fluorescence immune chromatography kit is configured to jointly with Wash buffer solutions.
3) detection of sample
A) HER2 antigen standards are formulated as 2ng/mL by the use of normal human serum as dilution respectively and 350ng/mL is dense
Degree；
B) by the HER2 standard solutions of two kinds of concentration in step a) successively according to 100:0、92:8、75:25、50:50、
25:75 and 0:100 ratio is mixed；
C) serum solution (100 μ L) prepared in step b) is added dropwise in well (9) respectively, forward direction chromatography reaction
15min；
D) Wash buffer solutions, pH value 7.5 are prepared, buffer system is 20mM phosphate, and it is (dense to add bovine serum albumin(BSA)
Spend for 1%), sucrose (concentration 10%) and surfactant triton x-100 (concentration 0.8%), added in sample well
50 μ L, stand 5min.
E) it is placed in fluorescent quantitation instrument and obtains fluorescence signal intensity, and draw corresponding standard curve, refers to Fig. 4；
F) operated with step c) and step d), testing sample is detected, Fig. 3 is shown at luminoscope detection peak, and chromatography terminates
Afterwards, it is placed in fluorescent quantitation instrument and obtains fluorescence signal intensity, and HER2 in the sample is analyzed according to the standard curve in step e)
Content；
G) examining report is exported.
4) interpretation of result
As a result show, its lowest detection is limited to 0.5ng/mL, minimum to be quantitatively limited to 2ng/mL, and batch in batch between it is repeated
Preferably, coefficient R 2>0.99.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (8)
1. a kind of two-photon fluorescence immune chromatography reagent kit for quantitatively detecting human epidermal growth factor receptor 2 (HER2), including buckle
(11), two-photon fluorescence immuno-chromatographic test paper strip and Wash buffer solutions, it is characterised in that：
Buckle (11) is the outside shell structure of fluorescence immune chromatography test paper bar, including well (9) and observation window (10)；
Fluorescence immune chromatography test paper bar includes sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and bottom plate (8), sample
Product pad (1), label pad (2), chromatographic film (6) and adsorptive pads (7) are equipped on bottom plate (8), and sample pad (1) is located at well
(9) lower section, and label pad (2) is connected to, label pad (2) is connected to chromatographic film (6), and chromatographic film (6) is connected to adsorptive pads
(7) a quantitative detection line (4) and a nature controlling line (5), detection line (4) and nature controlling line (5) position, are fixed with chromatographic film (6)
In the lower section of observation window (10)；
Human epidermal growth factor receptor 2 (HER2) specific antibody of fluorescent dye modification is fixed with label pad (2) simultaneously, and it is glimmering
The Quality Control molecule of photoinitiator dye modification, the transmitted wave of the fluorescent dye of (HER2) specific antibody of modification human epidermal growth factor acceptor 2
The long launch wavelength with modifying the fluorescent dye of Quality Control molecule is identical, wave-length coverage 300-500nm；
Human epidermal growth factor receptor 2 (HER2) specific antibody of fluorescent dye modification, the spy are fixed with quantitative detection line (4)
The antigenic determinant and the human epidermal growth factor receptor for the fluorescent dye modification being fixed in label pad (2) that heterogenetic antibody is identified
The antigenic determinant that 2 (HER2) specific antibodies are identified is different；And be fixed with nature controlling line (5) can be with Quality Control molecular specific
Property the fluorescent dye modified biological molecule that combines, the fluorescence of (HER2) specific antibody of modification coating human epidermal growth factor receptor 2
The launch wavelength of fluorescent dye of the launch wavelength of dyestuff with modifying Quality Control molecule is identical, wave-length coverage 500-800nm；
2. kit according to claim 1, it is characterised in that the fluorescence immune chromatography test paper bar is also including blood point
From film (3), it is arranged between label pad (2) and chromatographic film (6), and now label pad (2) is connected to blood separation membrane (3), blood
Liquid seperation film (3) is connected to chromatographic film (6)；Or be arranged between sample pad (1) and label pad (2), now sample pad (1) connects
Blood separation membrane (3) is connected to, blood separation membrane (3) is connected to label pad (2)；Or directly merged into sample pad (1) same
Structure.
3. kit according to claim 1, it is characterised in that the mark fluorescent dyestuff is organic fluorescent dye, dilute
Earth elements fluorescent dye or quantum dot fluorescence dyestuff, launch wavelength 300-500nm, this example select 365nm.Fluorescent dye is to have
Machine fluorescent dye, rare earth element fluorescent dye or quantum dot fluorescence dyestuff, launch wavelength 500-800nm, this example choosing
635nm；And mark and coating fluorescent dye can exchange.Described one of dyestuff is rare earth element fluorescent dye, fluorescence
Microballoon or quantum dot fluorescence dyestuff；Another described dyestuff is that machine fluorescent dye is Alexa series near-infrared fluorescent chemical combination
At least one of thing, DyLight series near infrared fluorescent compound and CF series near infrared fluorescent compounds；Its feature exists
In the nearly organic fluorescent dye is the fluorescence of Alexa Fluro serial (Alexa Fluro647,610,633,700,750)
At least one of element, DyLight series, (DyLight649,633,549,680), CF647 etc..
4. kit according to claim 1, it is characterised in that the chromatographic film, bottom plate and buckle are when more than 550nm
Fluorescence is very weak or does not contain fluorescer, and preferably bottom plate is white, and surface has adhesive sticker, and both of which is free of fluorescer.
5. kit according to claim 1, it is characterised in that the Wash buffer solutions include bovine serum albumin(BSA), sugarcane
Sugar and surfactant, pH value 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is 0.05~2%, and the concentration of sucrose is 1
~15%, the concentration of surfactant is 0.05~2%.
6. the two-photon fluorescence immunochromatography according to claim 1 for quantitatively detecting human epidermal growth factor receptor 2 (HER2)
The preparation method of kit, it is characterised in that comprise the following steps：
Step (1)：With specific effect between chemical crosslinking or biomolecule respectively by conjugated fluorescent dyes to human epidermal growth factor
The surface of (HER2) specific antibody of acceptor 2 and Quality Control molecule, respectively obtain the human epidermal growth factor receptor 2 of fluorescent dye modification
(HER2) specific antibody and the Quality Control molecule of fluorescent dye modification, the launch wavelength of the fluorescent dye of the Quality Control molecule is modified
The launch wavelength of fluorescent dye with modifying human epidermal growth factor acceptor 2 (HER2) specific antibody is identical, wave-length coverage 300
~500nm；Coating conjugated fluorescent dyes are given birth to coating people's epidermis respectively with specific effect between chemical crosslinking or biomolecule
The surface of long (HER2) specific antibody of factor receptor 2 and Quality Control molecule, respectively obtain the coating people epidermis life of fluorescent dye modification
The Quality Control molecule of long (HER2) specific antibody of factor receptor 2 and fluorescent dye modification, modify the fluorescent dye of the Quality Control molecule
Launch wavelength with modify human epidermal growth factor acceptor 2 (HER2) specific antibody fluorescent dye launch wavelength it is identical, ripple
Long scope is 500~800nm；
Step (2)：(HER2) specific antibody of human epidermal growth factor receptor 2 that the fluorescent dye that step (1) is obtained is modified and
The Quality Control molecule of fluorescent dye modification is fixed in label pad (2)；
Step (3)：One quantitative detection line (4) and a nature controlling line (5) are set respectively in chromatographic film (6), wherein, nature controlling line
(5) biomolecule for the fluorochrome label that can be combined with Quality Control molecular specificity is fixed with, it is fixed in quantitative detection line (4)
There are human epidermal growth factor receptor 2 (HER2) specific coated antibody of fluorochrome label, and the specific fluorescent dye marker
The antigenic determinant and the human epidermal growth factor receptor for the fluorescent dye modification being fixed in label pad (2) that coated antibody is identified
The antigenic determinant that 2 (HER2) specific antibodies are identified is different；
Step (4)：Sample pad (1), label pad (2), chromatographic film (6), adsorptive pads (7) and bottom plate (8) are built into fluorescence immunoassay
Chromatograph test strip；
Step (5)：Fluorescence immune chromatography test paper bar is loaded.
7. according to the method for claim 6, it is characterised in that the step of also including setting blood separation membrane (3), blood point
It is arranged on from film (3) between label pad (2) and chromatographic film (6), now label pad (2) is connected to blood separation membrane (3), blood point
Chromatographic film (6) is connected to from film (3)；Or be arranged between sample pad (1) and label pad (2), now sample pad (1) is connected to
Blood separation membrane (3), blood separation membrane (3) are connected to label pad (2)；Or directly merge into same structure with sample pad (1).
8. according to the method for claim 6, it is characterised in that the step of also including preparing Wash buffer solutions, Wash bufferings
Liquid includes bovine serum albumin(BSA), sucrose and surfactant, pH value 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is
0.05~2%, the concentration of sucrose is 1~15%, and the concentration of surfactant is 0.05~2%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710755819.2A CN107589264A (en) | 2017-08-31 | 2017-08-31 | Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710755819.2A CN107589264A (en) | 2017-08-31 | 2017-08-31 | Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107589264A true CN107589264A (en) | 2018-01-16 |
Family
ID=61050368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710755819.2A Pending CN107589264A (en) | 2017-08-31 | 2017-08-31 | Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107589264A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593923A (en) * | 2018-03-15 | 2018-09-28 | 福建医科大学附属第医院 | Application of the joint fluid neutrophil gelatinase-associated lipocalin in the detection of prosthetic joint infection and diagnostic kit |
| CN112816719A (en) * | 2020-12-25 | 2021-05-18 | 重庆康巨全弘生物科技有限公司 | POCT full-automatic fluorescence immunoassay appearance |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013120120A (en) * | 2011-12-07 | 2013-06-17 | Konica Minolta Medical & Graphic Inc | Test strip for lateral flow type chromatography, and method for detecting or quantifying analyte using the same |
| CN104655858A (en) * | 2015-03-12 | 2015-05-27 | 德迈基生物技术（北京）有限公司 | Fluorescence immunochromatography kit for quantitatively detecting human epididymis secretory protein-4 and preparation method for fluorescence immunochromatography kit |
| CN105238388A (en) * | 2015-09-21 | 2016-01-13 | 遵义师范学院 | Two-photon fluorescent probe, fluorescence-labeled kit and detection method used for biopsy of intracellular carbohydrate chain antigen |
| CN105403704A (en) * | 2015-12-31 | 2016-03-16 | 苏州市博纳泰科生物技术有限公司 | Fluorescence immunoassay detection method and kit for HER-2 |
| CN106124757A (en) * | 2011-11-21 | 2016-11-16 | 爱贝斯股份有限公司 | Signal during lateral flow and related immune measure amplifies |
-
2017
- 2017-08-31 CN CN201710755819.2A patent/CN107589264A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106124757A (en) * | 2011-11-21 | 2016-11-16 | 爱贝斯股份有限公司 | Signal during lateral flow and related immune measure amplifies |
| JP2013120120A (en) * | 2011-12-07 | 2013-06-17 | Konica Minolta Medical & Graphic Inc | Test strip for lateral flow type chromatography, and method for detecting or quantifying analyte using the same |
| CN104655858A (en) * | 2015-03-12 | 2015-05-27 | 德迈基生物技术（北京）有限公司 | Fluorescence immunochromatography kit for quantitatively detecting human epididymis secretory protein-4 and preparation method for fluorescence immunochromatography kit |
| CN105238388A (en) * | 2015-09-21 | 2016-01-13 | 遵义师范学院 | Two-photon fluorescent probe, fluorescence-labeled kit and detection method used for biopsy of intracellular carbohydrate chain antigen |
| CN105403704A (en) * | 2015-12-31 | 2016-03-16 | 苏州市博纳泰科生物技术有限公司 | Fluorescence immunoassay detection method and kit for HER-2 |
Non-Patent Citations (1)
| Title |
|---|
| 刘玲芝: "双光子激发—荧光共振能量转移及其生物分析应用", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593923A (en) * | 2018-03-15 | 2018-09-28 | 福建医科大学附属第医院 | Application of the joint fluid neutrophil gelatinase-associated lipocalin in the detection of prosthetic joint infection and diagnostic kit |
| CN112816719A (en) * | 2020-12-25 | 2021-05-18 | 重庆康巨全弘生物科技有限公司 | POCT full-automatic fluorescence immunoassay appearance |
| CN112816719B (en) * | 2020-12-25 | 2023-12-12 | 重庆康巨全弘生物科技有限公司 | POCT full-automatic fluorescence immunoassay appearance |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104655858B (en) | Fluorescence immune chromatography kit of quantitative detection people epididymal secretory protein-4 and preparation method thereof | |
| CN107621540A (en) | Quantitatively detect anti-Miao Le pipes hormone（AMH）Two-photon fluorescence immune chromatography reagent kit and preparation method thereof | |
| CN106771207A (en) | A kind of cancer of pancreas dry type fluorescence quantum joint inspection diagnostic kit | |
| CA2363566A1 (en) | Method of diagnosing and monitoring malignant breast carcinomas | |
| CN1362970A (en) | Monoclonal antibody against estrogen-stimulated leucine aminopeptidase | |
| US20190187154A1 (en) | Biomedical measuring devices, systems, and methods for measuring peptide concentration to monitor a condition | |
| CN204514928U (en) | Detect the test strips of pepsinogen I and II and apply its test card | |
| CN109975559B (en) | A kit and method for time-resolved fluorescence quantitative detection of 25-hydroxyvitamin D | |
| CN107389928A (en) | Quantitatively detect two-photon fluorescence immune chromatography reagent kit of gastrin-releasing peptide precursor (pro GRP) and preparation method thereof | |
| Mukundan et al. | Planar optical waveguide-based biosensor for the quantitative detection of tumor markers | |
| WO2018068582A1 (en) | Immunochromatography detection apparatus | |
| CN109142758A (en) | It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof | |
| CN105527440A (en) | Immunochromatographic test paper strip and preparation method and application thereof | |
| JP2012247188A (en) | Clinical examination using nanocarbon | |
| CN101344526A (en) | Fast and accurate prostatic cancer detection test paper strip and its preparation and application | |
| CN106596937A (en) | Quantum dot kit for synchronous detection of pancreatic cancer marker | |
| CN108872438A (en) | Lung cancer marker GK5 quick detection kit in a kind of excretion body | |
| CN107589264A (en) | Quantitatively detect human epidermal growth factor receptor 2（HER2）Double light ancestral fluorescence immune chromatography kits and preparation method thereof | |
| CN109444422A (en) | A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II | |
| JP6998626B2 (en) | An immunological composition for diagnosing lung cancer using an autoantibody-antigen conjugate, a method for diagnosing lung cancer using the same, and a kit for diagnosing lung cancer containing the same. | |
| EP3550304B1 (en) | Method for estimating gleason score of prostate cancer, method for estimating pathological stage, and method for acquiring supplementary information, all on the basis of specific psa content in specimen | |
| CN109116023B (en) | Lung cancer marker anti-MMP 12 autoantibody and application thereof | |
| CN101551387A (en) | Immunohistochemical analysis and detection method based on quantum dots | |
| EP3358352B1 (en) | Method for estimating pathological tissue diagnosis result (gleason score) of prostate cancer | |
| CN103712963B (en) | A kind of fluorescence analysis method and device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180116 |