CN109884316A - It is a kind of for detecting the kit and preparation method of tumor M 2-type pyruvate kinase - Google Patents
It is a kind of for detecting the kit and preparation method of tumor M 2-type pyruvate kinase Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the kit and preparation method of tumor M 2-type pyruvate kinase, kit includes test card, test card includes bottom plate, bottom plate is equipped with the sequentially connected sample pad in end, bonding pad, nitrocellulose membrane and water absorption pad along sample flow direction, bonding pad is adsorbed with quantum dot-labeled capture antibody, nitrocellulose membrane is successively arranged detection band and quality control band along sample flow direction, and detection band is coated with detection antibody, and quality control band is coated with IgG；Capture antibody, detection antibody is antitumor M2 type pyruvate kinase antibody, the different epitopes of capture antibody tumor M 2-type pyruvate kinase in conjunction with detection antibody；Antibody is captured before marking quantum dot, using the sugar-modified capture antibody of monophosphate.Kit of the present invention has high sensitivity, specificity, and operation is fast and convenient, result is accurate, economic and practical.
Description
Technical field
The invention belongs to vitro diagnostic techniques fields more particularly to a kind of for detecting the examination of tumor M 2-type pyruvate kinase
Agent box and preparation method.
Background technique
Colorectal cancer is one of three big malignant tumours common in the world, and the death rate ranked third position.In recent years, with people
Improvement of living standard and the change of dietary structure, disease incidence be in obvious ascendant trend.But at present both at home and abroad to knot
The early diagnosis of the carcinoma of the rectum has entered mid-term and advanced colorectal cancer when about half patient clarifies a diagnosis still in reduced levels.
Therefore, the early diagnosis for improving colorectal cancer is current domestic and international critical issue in the urgent need to address.Early diagnosis knot at present
The carcinoma of the rectum has following several common technology: (1) FOB Fecal Occult Blood Testing is the common screening method of colorectal cancer, easy to operate,
Noninvasive, major defect is that sensitivity is low, poor specificity, and false positive results are high；(2) electronic colonoscope (abbreviation scope) be discovery and
The most effective means of colorectal cancer are diagnosed, and the main method of early diagnosis colorectal cancer at present, especially in people at highest risk
Inspection have important clinical value；Because it with the side effect of the danger such as bleeding, enterobrosis and is needed for traumatic examination
Certain equipment and instrument, this is more demanding to the professional standards of operator, therefore there is very in the inspection of a wide range of crowd
Big limitation；(3) Electrochemiluminescence technology detects common tumor markers, such as CEA, CA199, CA242, CA50, Yin Qiyang
Property rate it is low, lack specificity, the improper early diagnosis for colorectal cancer；(4) using round pcr detection patients serum or excrement
Just the concentration of DNA and detection Microrna in are monitored to treatment of colorectal cancer and prognostic evaluation has certain value, but they
It needs further to study in terms of early diagnosis applied to colorectal cancer.In recent years, domestic and foreign scholars start to be dedicated to seeking
It looks for sensitive, special, reliable, effective colorectal cancer neoformation marker and establishes noninvasive testing and experimental method to mention
The diagnosis of height early diagnosis colorectal cancer.Foreign scholar thinks tumour M2Type pyruvate kinase (M2- PK) it is examined in blood plasma, excrement
It is higher to survey its sensitivity and specificity, is expected to become colorectal cancer novel tumor markers.
Pyruvate kinase has 4 kinds of different isodynamic enzymes, respectively L-type, R type, M1 type, M2 type, the distribution of this 4 kinds of isodynamic enzymes
With tissue specificity.M2-PK is distributed mainly on lung tissue, distal convoluted renal tubule, embryonic tissue and proliferation or undifferentiated group
It is to exist in knitting, under normal cell activated state with the tetramer of high activity.In tumour cell, have proven to due to junket ammonia
Acid acceptor phosphorylation causes tetramer type to crack, and is changed into and mainly exists in the form of dimer, and great expression, therefore, claims
The patient for seeing most of malignant tumours is increased for tumor M2-PK, tumor M2-PK.Although from the swollen of different tissues
The level of the tumour of tumor and different stages, tumor M2-PK expression has difference, but tumor M2-PK is as tumor-marker
Object is that have very much application value.Tumor M2-PK is a kind of more novel tumor markers of Recent study, pernicious swollen
Preferable application prospect is shown in the early diagnosis of tumor and judging prognosis.
Mankind's Colon and rectum epithelium renewal speed quickly, is about updated with per hour 1% speed, there is nearly 10 daily10On normal
Chrotoplast falls off.1 contains 109If the 1cm size tumor tissues of cell are updated with normal speed, fall off into the knot of enteron aisle
At least 1% tissue from the tumour in rectum epithelial cell total amount.Tumour cell falls off into enteric cavity as body is discharged in excrement
Outside, the detection for cast-off cells mutated gene and its expression product in excrement is laid a good foundation.Tumour towards enteric cavity is to enteric cavity
Endocrine metabolism product is largely decomposed with the distinctive substance of certain tumours, and the tumour cell to fall off, discharges intracellular matter,
And tumor markers are avoided by excrement and are decomposed by liver metabolism so that by excrement detect tumor markers can
Row.Therefore, colorectal cancer is diagnosed by tumor M2-PK level in detection excrement is a kind of objective, reliable experimental method.
Early in 1993, in German 7th tumor markers seminar, tumor M2-PK just swelled as a kind of new
Tumor markers are suggested.Applied tumor M2-PK kit and stool latent every year in the several countries in the Europe such as Germany later
Auxiliary diagnosis of the blood as the early screening of colorectal cancer.In recent years, scholar carries out tumor M2-PK both at home and abroad
Research, is shown in the diagnosis of colorectal cancer, tumor M2-PK has using the enzyme linked immunological method (ELISA) of import Germany
Higher sensibility and specificity, difference 73% and 78%, they think that the tumor M2-PK in excrement is a kind of ideal
Colorectal carcinoma marker.Currently, external scholar thinks, the sensibility of the tumor M2-PK in excrement is higher than in serum,
It may be tumour to blood circulation and enteric cavity while under the premise of secrete tumor M2-PK, the tumour cell of tumor surface necrosis
Release tumor M2-PK is decomposed, and when tumour does not grow into certain degree, the tumor of tumors secrete to blood circulation
M2-PK amount is smaller, and the amount of the tumor M2-PK in enteric cavity is more than the tumor M2-PK amount of blood circulation, this causes in excrement
Detect that the content of tumor M2-PK is apparently higher than blood circulation.
Chinese invention patent 200610061296.3 discloses human tumor M 2-type pyruvate kinase antigen determinant polypeptide, resists
Body and its application on diagnostic kit, by the way that the antigen polypeptide of two kinds of separate sources is prepared two kinds of different epitopes
Tumor M2-PK polyclonal antibody, it is one such to be used as coated antibody；Tumor M2-PK and ELISA Plate is coated in sample
Upper tumor M2-PK specific polyclonal antibody combines, in addition incubating 30 minutes, board-washing believes fluorescence after enzyme mark secondary antibody
It number is fixed on ELISA Plate, the concentration of tumor M2-PK is determined by fluorescence microplate reader fluorescence intensity.However this method is examined
The coating and closing, the processing of standard items or tested serum, polyclonal antibody detection that ELISA Plate is needed when survey, in detection process
Brooding time length, board-washing often, need mating professional equipment and professional.
The chemiluminescence that Chinese invention patent CN106405094A discloses for detecting knubble type M 2 pyruvate kinase is exempted from
Epidemic disease kit, it includes reagent have tumor M2-PK Magneto separate reagent, enzyme label tumor
M2-PK antibody-solutions, standard items, cleaning solution and chemiluminescent substrate solution (are divided into luminous substrate solution A and hair
Light substrate B solution), the Tris-HCl buffer that luminous substrate A is 0.1M, pH value is 8.5, and contain final concentration in the buffer
For 5.0mg/mL luminol, luminous substrate B be 0.1M citrate buffer solution that pH value is 4.5, and containing eventually in the buffer
Concentration is the hydrogen peroxide and 15mg/mL horseradish peroxidase of 100mg/mL；It is realized using the principle of double antibody sandwich method
Quantitative detection tumor M2-PK.However this method operationally needs to be equipped with expensive chemiluminescent analyzer and professional,
It is only capable of detection serum or plasma sample in party's science of law, the secretion including urine and excrement cannot be detected.
Therefore, how to develop short detection time, easy to operate, the accurate tumor M2-PK detection method of testing result and
Testing product becomes urgent problem to be solved.
The tumor M2-PK kit developed both at home and abroad at present is all made of enzyme immunization method, and this method detecting step is more, behaviour
The influence factor for making process generation is more, and testing result easily causes deviation, and time and effort consuming.Although quanta point biological marks
For aspect it has been reported that still, quantum point coupling antibody and purifying but have that complicated for operation, the rate of recovery is low, is difficult to realize batch examines
The drawbacks such as survey and scale.Therefore, how technology of quantum dots to be applied in tumor M 2-type pyruvate kinase simultaneously efficient, simplicity
Detection is a urgent problem.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and provide with one kind for detecting tumor M 2-type acetone
The kit and preparation method of acid kinase, using the surface of monophosphate sugar allosteric activation capture antibody, enhancing captures anti-the present invention
Body for the first time catches quantum dot-labeled with coupling tumor M 2-type pyruvate kinase, kit provided by the present invention in conjunction with quantum dot
It obtains antibody to be applied in tumor M 2-type pyruvate kinase detection, which has high sensitivity, specificity, and operates quickly letter
Just, result is accurate, economic and practical.
To achieve the above object, the technical scheme adopted by the invention is as follows: one kind is for detecting tumor M 2-type pyruvate kinase
Test card, the test card includes bottom plate, and bottom plate is equipped with the sequentially connected sample pad in end, combination along sample flow direction
Pad, nitrocellulose membrane and water absorption pad, the bonding pad are adsorbed with quantum dot-labeled capture antibody, and nitrocellulose membrane is along sample flow
Dynamic direction is successively arranged detection band and quality control band, the detection band are coated with detection antibody, and it is (excellent that the quality control band is coated with IgG
It is selected as sheep anti-mouse igg)；
The capture antibody is antitumor M2 type pyruvate kinase antibody, and the capture antibody is adopted before marking quantum dot
With the surface of the sugar-modified capture antibody of monophosphate；The detection antibody is antitumor M2 type pyruvate kinase antibody, the capture
The different epitopes of antibody tumor M 2-type pyruvate kinase in conjunction with detection antibody；
The sugar-modified capture antibody of the monophosphate is to be prepared by the following method: single phosphorus being added into capture antibody
Sour sugar and bovine serum albumin(BSA) buffer, are placed at room temperature for 1~2h after shaking up；The pH value of solution is adjusted to 8~11 again；It is excellent
200~500mg monophosphate sugar and 200~800mg/mL bovine serum albumin(BSA) is added into 300~800mg capture antibody in selection of land
Buffer places 1~2h of room temperature after shaking up, be ready for activation quantum dot is added: after monophosphate sugar is added in capture antibody,
The N-terminal area the glycosylation modified Ji Fc generation for capturing antibody is glycosylation modified, on the one hand makes the N-terminal increased activity of antibody, with
The carboxyl of quantum dot combines；On the other hand one or more aldehyde functional group, the acyl of aldehyde radical and quantum dot are generated on the surface of antibody
Hydrazine reaction is coupled, and stability is greatly increased.
Preferably, capture antibody described above and detection antibody are that recombined human tumor M2-PK antigen is passed through cell gram
The grand hybridoma cell strain for obtaining two plants of secretory antibodies, is 33.26 hybridoma cell strain of M2 type pyruvate kinase and M2 type respectively
31.3 hybridoma cell strain of pyruvate kinase；The genetic recombination tumor M2-PK albumen is using carrying tumor M2-PK egg
A kind of engineered strain preparation of Escherichia coli of white gene, preparation carries out according to a conventional method.
In addition, the present invention also provides the preparation methods of the test card comprising following steps: the bonding pad presses 1
~3 μ L/cm spray the quantum dot-labeled capture antibody-solutions of 0.1~5mg/mL, and the detection band is by 1~3 μ L/cm spraying 0.1
~5mg/mL detects antibody-solutions, and the quality control band sprays 0.1~5mg/mL IgG solution by 1~3 μ L/cm；It is quantum dot-labeled
Capture antibody-solutions, detection antibody-solutions and IgG solution solvent be coating buffer, the coating buffer is includes 20
~150mM NaCl, 0.05%~3%PEG (w/v), 0.2%~1% trehalose (w/v), 2~10mg/mL BSA (w/v),
0.05%NaN310~100mM PBS buffer solution.
As an improvement of the above technical solution, the quantum dot-labeled capture antibody is through the following steps that made
It is standby:
S1) quantum point activation: use N- hydroxy thiosuccinimide (NHS) by the carboxyl of CdSe/ZnS quantum dot surface
It activates (carboxyl water-soluble CdSe/ZnS quantum dot)；Next in reaction, in ethyl -3- carbodiimides hydrochloride (EDC)
Under the action of, the carboxyl of the quantum dot of activation will form amido bond with the original amino of capture antibody surface；
S2) using the surface of the sugar-modified capture antibody of monophosphate to get the capture antibody of activation；Quantum dot and capture antibody
Before success is coupled, by the effect of application monophosphate sugar allosteric activation, on the one hand the surface texture of modification capture antibody is improved
The activity of antibody is captured, on the other hand the function of enhancing capture antibody N-terminal structure, is conducive in conjunction with quantum dot and is coupled
Tumor M 2-type pyruvate kinase；
S3) quantum dot of activation is placed to room temperature, ethyl -3- carbodiimides hydrochloric acid is added into the quantum dot of activation
Compound solution, N- hydroxy thiosuccinimide solution, BSA solution, are added the capture antibody of the activation, after shaking up after shaking up
It is incubated for, it is molten that ethyl -3- carbodiimides hydrochloride solution, N- hydroxy thiosuccinimide is added during being incubated for
Liquid；Methanol is added after incubation, and oscillation is protected from light after mixing；It adds beta -mercaptoethanol to be terminated, be used after terminating reaction
Beta -mercaptoethanol is dialysed；Centrifugation removal supernatant is after dialysis to get quantum dot-labeled capture antibody.
As a further improvement of the above technical scheme, in step sl, the concentration of the quantum dot of activation is 1~10 μM；
In step s 2, the concentration of the capture antibody is 10~100 μ g/ml；In step s3, the concentration of the BSA solution is 20
The mass ratio of~200mg/mL, the capture antibody of the quantum dot and activation of activation are (1:2)~(1:10).
It is further improved as above-mentioned technical proposal, in step s3, the quantum dot of activation and the capture of activation are anti-
When body is coupled, the pH value of solution is 9~11, and the concentration of ethyl -3- carbodiimides hydrochloride solution is 9.38% (w/
V), the concentration of N- hydroxy thiosuccinimide solution is 10% (w/v), the quantum dot of activation and the ethyl -3- being added twice
The mass ratio of carbodiimides hydrochloride is 5~10, and the quantum dot of activation and the N- hydroxy succinyl being added twice are sub-
The mass ratio of amine is 5~10.
As an improvement of the above technical solution, the bonding pad is quantum dot-labeled by 2 μ L/cm spraying, 0.1~5mg/mL
Antibody-solutions are captured, the detection band sprays 0.1~5mg/mL by 2 μ L/cm and detects antibody-solutions, and the quality control band presses 2 μ L/cm
Spray 0.1~5mg/mL IgG solution.
Preferably, it detects and is divided into 5~7mm between band and control band, after spraying, be placed on 30%, 35~37 DEG C of humidity <
Under the conditions of dry for 24 hours after, be placed in spare in constant temperature and humidity safe.
As an improvement of the above technical solution, the sample pad is impregnated using pretreatment fluid, then is dried；The combination
Pad is impregnated, then dry before spraying using pretreatment fluid；The pretreatment fluid is containing 0.02%~0.1%Tween-20
(w/v) 0.01M and PH is 7~7.3PBS buffer；The pretreatment fluid has good adaptability to a variety of antigens and antibody.
Preferably, sample pad and the pretreated step of bonding pad are as follows: impregnate 1~2h in pretreatment fluid, be put in 36 after taking-up
It is saved backup after~38 DEG C of drying or 37 DEG C of vacuum freeze dryings.Water absorption pad uses absorbent wool；Bottom plate is using PVCJ glue
Plate plays and sample pad, bonding pad, nitrocellulose filter and water absorption pad links together.
In addition, the kit also includes to get stuck the present invention also provides a kind of kit comprising the test card, card
Shell includes back card and upper cover, and back card is equipped with card slot, and the test card is embedded in the card slot, the upper cover be equipped with testing window and
Well, the position of the testing window are matched with the position of the detection band and quality control band, the position of the well and institute
The position for stating sample pad matches.
As an improvement of the above technical solution, the kit also includes sample dissociating buffer, and the sample separation is slow
Fliud flushing is to state sample dissociating buffer to include medium, 0.45%~0.9% sodium chloride (w/v), 0.02%~0.1%
The PBS buffer solution of Tween-20 (w/v), 20~200mg/mL bovine serum albumin(BSA)；When the medium is cesium chloride, sample point
It is 10%~30% (v/v) from Cesium chloride concentrations in buffer；When the medium is sucrose, sucrose in sample dissociating buffer
Concentration is 0.1~0.5mol/L；When the medium is saccharoSan, saccharoSan concentration is 10% in sample dissociating buffer
~30% (w/v).
In addition, the present invention also provides the quantitative detecting methods of tumor M 2-type pyruvate kinase: with a series of various concentrations
Tumor M 2-type pyruvate kinase standard solution (more than 5 concentration), then using quantum dot quantitative fluorescence analysis instrument respectively into
The detection of row immunochromatography, by doing standard curve to each peak area corresponding concentration, then unknown sample to be checked is equally located
Reason, obtains peak area, learns tumor M 2-type pyruvate kinase content in sample according to calibration curve formula.
The invention has the advantages that: the present invention to provide a kind of kit for detecting tumor M 2-type pyruvate kinase and preparation
Method, the quantum dot-labeled combination fluorescence immune chromatography technology of first passage of the present invention swash to tumor M 2-type pyruvate in excrement
Enzyme is detected, and using the surface texture of the sugar-modified capture antibody of monophosphate before quantum dot-labeled capture antibody, is conducive to and is measured
Son point combines and coupling tumor M 2-type pyruvate kinase；The present invention is had the advantage that using quantum dot-labeled technology
1) its sensitivity and specificity is superior to other organic fluorescences label, such as fluorescein or fluorescent microsphere, be due to
The unique superior optical characteristics of quantum dot are determined there is very strong fluorescence intensity and longer fluorescence lifetime；
Quantum dot-labeled technology is combined with immunochromatography technique, by the variation of fluorescence intensity come the antigenic content in response sample,
Realize the quantitative detection to sample；Existing colloidal gold strip can only qualitative detection；
2) specificity and sensitivity of detection, testing result visitor testing result specificity and high sensitivity: are greatly improved
See and it is sensitive, sensitivity can reach 100~1000 times of number of traditional colloidal gold strip；
Early stage auxiliary diagnosis of the detection kit of the present invention in CRC has broad application prospects, and is especially suitable in base
In the use of layer hospital and the physical examination sieving and diagnosis of middle people at highest risk.
Detailed description of the invention
Fig. 1 is the axonometric drawing of test card of the present invention；Wherein, 1, bottom plate, 2, sample pad, 3, bonding pad, 4, detection band, 5, matter
Control band, 6, nitrocellulose membrane, 7, water absorption pad, 8, chromatography direction；
Fig. 2 is the forming process of the quantum dot-labeled capture antibody of display.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiments and the drawings pair
The present invention is described further.
The present invention provides a kind of test card (as shown in Figure 1) for detecting tumor M 2-type pyruvate kinase, the test paper
Card includes bottom plate, and bottom plate is equipped with the sequentially connected sample pad in end, bonding pad, nitrocellulose membrane and water suction along sample flow direction
Pad, the bonding pad are adsorbed with quantum dot-labeled capture antibody, and nitrocellulose membrane is successively arranged detection along sample flow direction
Band and quality control band, the detection band are coated with detection antibody, and the quality control band is coated with IgG；
The capture antibody is antitumor M2 type pyruvate kinase antibody, and the capture antibody is adopted before marking quantum dot
With the surface of the sugar-modified capture antibody of monophosphate；The detection antibody is antitumor M2 type pyruvate kinase antibody, the capture
The different epitopes of antibody tumor M 2-type pyruvate kinase in conjunction with detection antibody.
Preferably, the reagent card is prepared by following steps: the bonding pad by 1~3 μ L/cm spraying 0.1~
5mg/mL quantum dot-labeled capture antibody-solutions, the detection band are molten by 1~3 μ L/cm spraying 0.1~5mg/mL detection antibody
Liquid, the quality control band spray 0.1~5mg/mL IgG solution by 1~3 μ L/cm；Quantum dot-labeled capture antibody-solutions, detection
The solvent of antibody-solutions and IgG solution is coating buffer, and the coating buffer is to include 20~150mM NaCl, 0.05%
~3%PEG (w/v), 0.2%~1% trehalose (w/v), 2~10mg/mL BSA (w/v), 0.05%NaN310~100mM
PBS buffer solution.
Preferably, the quantum dot-labeled capture antibody is through the following steps that prepared (as shown in Figure 2):
S1) quantum point activation: using N- hydroxy thiosuccinimide by the activated carboxylic of CdSe/ZnS quantum dot surface；
S2 it) uses the sugar-modified capture antibody of monophosphate: monophosphate sugar being added into capture antibody and bovine serum albumin(BSA) is slow
Fliud flushing is placed at room temperature for 1~2h after shaking up；The pH value of solution is adjusted to the capture antibody to 8~11 to get activation again；
S3) quantum dot of activation is placed to room temperature, ethyl -3- carbodiimides hydrochloric acid is added into the quantum dot of activation
Compound solution, N- hydroxy thiosuccinimide solution, BSA solution, are added the capture antibody of the activation, after shaking up after shaking up
It is incubated for, it is molten that ethyl -3- carbodiimides hydrochloride solution, N- hydroxy thiosuccinimide is added during being incubated for
Liquid；Methanol is added after incubation, and oscillation is protected from light after mixing；It adds beta -mercaptoethanol to be terminated, be used after terminating reaction
Beta -mercaptoethanol is dialysed；Centrifugation removal supernatant is after dialysis to get quantum dot-labeled capture antibody.
Preferably, in step sl, the concentration of the quantum dot of activation is 1~10 μM；In step s 2, the capture antibody
Concentration be 10~100 μ g/ml；In step s3, the concentration of the BSA solution is 20~200mg/mL, the quantum dot of activation
Mass ratio with the capture antibody of activation is (1:2)~(1:10).
It is highly preferred that in step s3, when the quantum dot of activation and the capture antibody of activation are coupled, the pH value of solution
It is 9~11, the concentration of ethyl -3- carbodiimides hydrochloride solution is 9.38% (w/v), N- hydroxy thiosuccinimide
The concentration of solution is 10% (w/v), the quality of the quantum dot of activation and the ethyl -3- carbodiimides hydrochloride being added twice
Than being 5~10, the mass ratio of the quantum dot of activation and the N- hydroxy thiosuccinimide being added twice is 5~10.
Preferably, the bonding pad sprays the quantum dot-labeled capture antibody-solutions of 0.1~5mg/mL by 2 μ L/cm, described
Detection band sprays 0.1~5mg/mL by 2 μ L/cm and detects antibody-solutions, and the quality control band sprays 0.1~5mg/ by 1~2 μ L/cm
ML IgG solution.
Preferably, the sample pad is impregnated using pretreatment fluid, then is dried；The bonding pad is used before spraying
Pretreatment fluid is impregnated, then is dried；The pretreatment fluid is 0.01M and PH containing 0.02%~0.1%Tween-20 (w/v)
For 7~7.3PBS buffer.
In addition, kit also includes to get stuck the present invention also provides a kind of kit comprising the test card, get stuck packet
Back card and upper cover are included, back card is equipped with card slot, and the test card is embedded in the card slot, and the upper cover is equipped with testing window and sample-adding
Hole, the position of the testing window are matched with the position of the detection band and quality control band, the position of the well and the sample
The position of product pad matches.
Preferably, the kit also includes sample dissociating buffer, the sample dissociating buffer be include medium,
0.45%~0.9% sodium chloride (w/v), 0.02%~0.1%Tween-20 (w/v), 20~200mg/mL bovine serum albumin(BSA)
PBS buffer solution；When the medium is cesium chloride, Cesium chloride concentrations are 10%~30% (v/v) in sample dissociating buffer；
When the medium is sucrose, sucrose concentration is 0.1~0.5mol/L in sample dissociating buffer；When the medium is poly sugarcane
When sugared, saccharoSan concentration is 10%~30% (w/v) in sample dissociating buffer.
Embodiment 1
The present embodiment provides the preparation methods of quantum dot-labeled capture antibody comprising following steps:
S1) quantum point activation: using N- hydroxy thiosuccinimide by the activated carboxylic of CdSe/ZnS quantum dot surface；
S2) using the sugar-modified capture antibody of monophosphate to get the capture antibody of activation；
It S3 is) that carboxyl water-soluble CdSe/ZnS (1~10 μM) quantum dot is balanced to room temperature by concentration, into activation quantum dot
EDC solution (9.38%), NHS solution (10%) and BSA solution (20~200mg/mL) is added, the capture of activation is added after shaking up
Antibody (10~100 μ g/ml) after shaking up and is incubated for 30~45min, and addition EDC solution (9.38%), NHS are molten during being incubated for
Liquid (10%)；Methanol is added after incubation, mixing is protected from light 1.5~2h of concussion；It adds beta -mercaptoethanol to be terminated, terminate
Beta -mercaptoethanol is added after reaction and stablizes quantum dot, is dialysed；It is centrifuged 3min under the conditions of 15000g after dialysis, is removed
Supernatant；Precipitating (i.e. quantum dot-labeled capture antibody) is resuspended in PBS, 4 DEG C of preservations；
Wherein, in step s3, when the quantum dot of activation and the capture antibody of activation are coupled, the pH value of solution is 9,
The mass ratio of the quantum dot of activation and the ethyl -3- carbodiimides hydrochloride being added twice is 5, the quantum dot of activation and two
The mass ratio of the N- hydroxy thiosuccinimide of secondary addition is 5；The mass ratio of the capture antibody of the quantum dot and activation of activation
For 1:2.
Embodiment 2
The present embodiment provides the preparation methods of quantum dot-labeled capture antibody, are with the difference of embodiment 1: in step
In S3, when the quantum dot of activation and the capture antibody of activation are coupled, the pH value of solution is 10, the quantum dot of activation with twice
The mass ratio of the ethyl -3- carbodiimides hydrochloride of addition is 7, the quantum dot of activation and the N- hydroxy being added twice
The mass ratio of succinimide is 8；The mass ratio of the capture antibody of the quantum dot and activation of activation is 1:7.
Embodiment 3
The present embodiment provides the preparation methods of quantum dot-labeled capture antibody, are with the difference of embodiment 1: in step
In S3, when the quantum dot of activation and the capture antibody of activation are coupled, the pH value of solution is 11, the quantum dot of activation with twice
The mass ratio of the ethyl -3- carbodiimides hydrochloride of addition is 10, the quantum dot of activation and the N- hydroxyl sulphur being added twice
Mass ratio for succinimide is 10；The mass ratio of the capture antibody of the quantum dot and activation of activation is 1:10.
Embodiment 4
The present embodiment provides the test card for detecting tumor M 2-type pyruvate kinase, test card includes bottom plate (PVC glue
Plate), bottom plate is equipped with the sequentially connected sample pad in end, bonding pad, nitrocellulose membrane and water absorption pad along sample flow direction, in conjunction with
Pad is adsorbed with quantum dot-labeled capture antibody, and nitrocellulose membrane is successively arranged detection band and quality control band along sample flow direction,
Detection band is coated with detection antibody, and quality control band is coated with sheep anti-mouse igg；It is anti-for antitumor M2 type pyruvate kinase to capture antibody
Body captures antibody before marking quantum dot, using the surface of the sugar-modified capture antibody of monophosphate；Detection antibody is antitumor M2 type
Pyruvate kinase antibody, the different epitopes of capture antibody tumor M 2-type pyruvate kinase in conjunction with detection antibody；
Sample pad is impregnated using pretreatment fluid, then is dried；Bonding pad is soaked before spraying using pretreatment fluid
Bubble, then dry；Pretreatment fluid is that 0.01M and PH containing 0.1%Tween-20 (w/v) are 7.3PBS buffer；
The bonding pad capture antibody-solutions quantum dot-labeled by 2 μ L/cm spraying 0.1mg/mL, the detection band press 2 μ L/cm
It sprays 0.1mg/mL and detects antibody-solutions, quality control band sprays 0.1mg/mL IgG solution by 2 μ L/cm；Quantum dot-labeled capture
The solvent of antibody-solutions, detection antibody-solutions and sheep anti-mouse igg solution is coating buffer, and coating buffer is to include 20mM
NaCl, 0.05%PEG (w/v), 0.2% trehalose (w/v), 2mg/mL BSA (w/v), 0.05%NaN310mM PBS buffering
Liquid.
Embodiment 5
The present embodiment provides the test cards for detecting tumor M 2-type pyruvate kinase, are with the difference of embodiment 4: 1)
The bonding pad capture antibody-solutions quantum dot-labeled by 1 μ L/cm spraying 5mg/mL, the detection band spray 5mg/mL by 1 μ L/cm
Antibody-solutions are detected, quality control band sprays 5mg/mL IgG solution by 1 μ L/cm；2) pretreatment fluid is containing 0.08%Tween-20 (w/
V) 0.01M and PH is 7PBS buffer；3) quantum dot-labeled capture antibody-solutions, detection antibody-solutions and sheep anti-mouse igg
The solvent of solution is coating buffer, and coating buffer is to include 130mM NaCl, 2%PEG (w/v), 0.8% trehalose (w/
V), 7mg/mL BSA (w/v), 0.05%NaN380mM PBS buffer solution.
Embodiment 6
The present embodiment provides the test cards for detecting tumor M 2-type pyruvate kinase, are with the difference of embodiment 4: 1)
The bonding pad capture antibody-solutions quantum dot-labeled by 3 μ L/cm spraying 0.1mg/mL, the detection band are sprayed by 3 μ L/cm
0.1mg/mL detects antibody-solutions, and quality control band sprays 0.1mg/mL IgG solution by 3 μ L/cm；2) pretreatment fluid is containing 0.02%
The 0.01M and PH of Tween-20 (w/v) is 7.2 PBS buffer solution；3) quantum dot-labeled capture antibody-solutions, detection antibody are molten
The solvent of liquid and sheep anti-mouse igg solution be coating buffer, coating buffer be comprising 150mM NaCl, 3%PEG (w/v),
1% trehalose (w/v), 10mg/mL BSA (w/v), 0.05%NaN3100mM PBS buffer solution.
Embodiment 7
The present embodiment provides a kind of kit comprising the test card for detecting tumor M 2-type pyruvate kinase, kits
Also comprising getting stuck, get stuck including back card and upper cover, back card is equipped with card slot, and test card is embedded in card slot, and upper cover is equipped with testing window
And well, the position of testing window are matched with the position of the detection band and quality control band, the position of well and the sample
The position of pad matches；Kit also includes sample dissociating buffer, and kit also includes sample dissociating buffer, sample separation
Buffer be include medium, 0.45% sodium chloride (w/v), 0.02%Tween-20 (w/v), 20mg/mL bovine serum albumin(BSA)
PBS buffer solution；Medium is cesium chloride, and Cesium chloride concentrations are 10%, 20% or 30% (v/v) in sample dissociating buffer.
Embodiment 8
The present embodiment provides a kind of kits comprising the test card for detecting tumor M 2-type pyruvate kinase, with reality
The difference for applying example 7 is: sample dissociating buffer be include medium, 0.6% sodium chloride (w/v), 0.08%Tween-20 (w/
V), the PBS buffer solution of 120mg/mL bovine serum albumin(BSA)；When medium is sucrose, sucrose concentration is in sample dissociating buffer
0.1,0.3 or 0.5mol/L.
Embodiment 9
The present embodiment provides a kind of kits comprising the test card for detecting tumor M 2-type pyruvate kinase, with reality
The difference for applying example 7 is: sample dissociating buffer be include medium, 0.9% sodium chloride (w/v), 0.1%Tween-20 (w/
V), the PBS buffer solution of 200mg/mL bovine serum albumin(BSA)；Medium is saccharoSan, and saccharoSan is dense in sample dissociating buffer
Degree is 10%, 20% or 30% (w/v).
Embodiment 10
The present embodiment provides the detection methods of tumor M 2-type pyruvate kinase in excrement comprising following steps:
1) it takes 10~15ml excrement cell dissociating buffer to mix with 5~7g excrement, is configured to mixed liquor, 10000 revs/min
Clock is centrifuged 10 minutes, is collected supernatant, is removed precipitated impurities part；It is spare to collect another test tube of supernatant placement；
2) in the well for taking 100 μ L supernatants to be added on test card, after reacting 5~10min, quantum dot fluorescence is utilized
Quantitative analysis instrument carries out immunochromatography detection respectively, by making standard curve to each peak area corresponding concentration, then by it is to be checked not
Know that sample is equally handled, obtain peak area, tumor M 2-type pyruvate kinase in sample is learnt according to calibration curve formula.
Kit performance measurement
The testing principle and method of kit
The kit of immunofluorescence chromatographic test paper based on quantum dot-labeled capture antibody, immunofluorescence chromatography reaction packet
Include two kinds of immunological methods of double antibody sandwich method and indirect method.Double antibody sandwich method is the reaction method for detecting band, determined antigen
Formed at bonding pad with quantum dot-labeled capture antibody immune complex (quantum dot-tumor M2-PK monoclonal antibody+
Tumor M2-PK antigen --- form immune complex), the compound carried out on nitrocellulose filter it is mobile and on film
Detection be combined with antibody (i.e. the anti-human tumor M2-PK monoclonal antibodies of different epitopes), formation it is cured be immunized it is compound
(i.e. the anti-human tumor M2-PK of quantum dot-tumor M2-PK monoclonal antibody+tumor M2-PK antigen+different epitopes is mono- for object
Clonal antibody --- form immune complex).Indirect method is the reaction method of quality control band, free quantum dot-tumor M2-PK
Monoclonal antibody will continue to chromatography movement, is captured and combines by the secondary antibody (sheep anti-mouse igg antibody) on quality control band, form solidification
Immune complex.
The sensitivity technique of kit
It is measured 20 times with zero standard's product (i.e. blank control is same, is free of albumen) as sample, it is equal to calculate its fluorescent value
Value and standard deviation.The resulting fluorescent value of standard deviation with the mean value of measured value plus 2 times substitutes into calibration curve equation y=
(2.8858x+271.42 R2=0.9985).The concentration being calculated is that its detection limit is substituted into result by above method
The detection limit that calibration curve equation is calculated is 0.1ng/ml, shows kit of the present invention and detection method to people
The detection of tumor M2-PK has the sensitivity of height.
In addition, as shown in table 1, kit immunochromatographic method of the present invention is straight in knot compared with enzyme-linked immunoassay method (ELISA)
The detection of intestinal cancer group excrement, 12.3~36.8ng/ml of kit results (normal reference value < 10ng/ of immunochromatographic method of the present invention
Ml), and ELISA the result is that 3.2~13.6ng/ml (the normal reference value < 6ng/ml of ELISA), the two comparing difference has system
Meter learns meaning (p < 0.01).Immunochromatographic method clinical detection result illustrates kit of the invention sensitivity with higher and very
Good accuracy, the result of non-false positive or false negative.And ELISA sensitivity is low, has 6 to be positive in 20 colorectal cancer patients
Often as a result, being clearly present higher false negative result.
The testing result (ng/ml) of tumor M2-PK in 1 excrement of table
The specific detection of kit
Tumor M2-PK detection kit of the present invention detects tumor M2-PK albumen and human serum in human faecal mass respectively
In tumor M2-PK albumen.Use with detection agent box of the invention detect respectively 10 parts of human faecal mass (5 parts for CRC patient, another 5
Part be people taking physical examination) in tumor M2-PK content (ng/ml), (5 parts are CRC patient, another 5 parts healthy bodies for 10 parts of serum
Inspection crowd) in tumor M2-PK content, in 10 parts of mouse excrement (5 parts are CRC animal model, another 5 parts of normal mouses)
Tumor M2-PK content in tumor M2-PK content and mouse serum (5 parts are CRC animal model, another 5 parts of normal mouses)
(ng/ml), testing result is shown in Table 2: in table 2, serial number 1~5 is people's CRC patient or mouse CRC patient, and serial number 6~10 is Healthy People
Group or normal mouse.
As shown in table 2, testing result show people's CRC patient excrement tumor M2-PK protein concentration be 18.9~
26.6ng/m, the excrement tumor M2-PK protein concentration of healthy population are 0.4~4.6ng/ml；The serum of people's CRC patient
Tumor M2-PK protein concentration is 10.4~13.6ng/ml, the serum tumor M2-PK protein concentration of healthy population is 0.3~
2.7ng/ml；The excrement tumor M2-PK protein concentration of mouse CRC animal model is 4.3~6.8ng/ml, the excrement of normal mouse
Tumor M2-PK protein concentration is 0.2~2.1ng/ml；The serum tumor M2-PK protein concentration of mouse CRC animal model is
3.7~5.3ng/ml, the serum tumor M2-PK protein concentration of normal mouse are 0.3~1.5ng/ml.It can be seen that this
Tumor M2-PK content in invention kit energy specific assay human faecal mass and serum.
Tumor M2-PK content (ng/ml) in 2 people of table, mouse excrement and serum
| Classification | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Excrement (people) | 26.6 | 19.2 | 21.2 | 18.9 | 19.9 | 4.6 | 4.2 | 3.7 | 2.3 | 0.4 |
| Serum (people) | 13.6 | 10.9 | 11.2 | 11.8 | 10.4 | 1.9 | 2.7 | 1.8 | 0.3 | 1.6 |
| Excrement (mouse) | 5.7 | 6.8 | 6.4 | 5.6 | 4.3 | 2.1 | 1.9 | 0.2 | 1.2 | 1.8 |
| Serum (mouse) | 4.3 | 5.3 | 5.1 | 4.4 | 3.7 | 1.5 | 1.8 | 0.9 | 1.1 | 0.3 |
The stability of kit
Three batches of reagents for making kit by oneself are respectively placed in 4 DEG C of half a year and 1 year, after 25~30 DEG C are placed 7~10 days,
Linear relationship and zero standard product fluorescence intensity level before comparing and placing between reference standard product each point fluorescence intensity level, detect each batch
Bright difference is not present with before placing as the result is shown in the stability that reagent closes.
The clinical application of kit
Detect clinical fecal sample and be divided into four groups: definitive pathological diagnosis is that colorectal cancer (CRC) organizes 20, and definitive pathological diagnosis is knot
Rectal polyp group 20, definitive pathological diagnosis is Colon and rectum adenoma group 20, and people taking physical examination is as Normal group 20.
As shown in table 3, testing result are as follows: in Normal group excrement the range of tumor M2-P content be 0.3~
2.4ng/ml；The range of tumor M2-PK content is 0.4~3.1ng/ml in colorectal polypus group excrement；Colon and rectum adenoma group
The range of tumor M2-PK content is 0.5~4.7ng/ml in excrement；The range of tumor M2-PK content is in CRC group excrement
12.3~36.8ng/ml；Comparing difference has statistical significance (p < 0.01).Therefore, above-mentioned clinical detection result illustrates this examination
Agent box sensitivity with higher and good accuracy, the result of non-false positive or false negative.
Tumor M2-PK content (ng/ml) in 3 each group excrement of table
In addition, inventing quantum dot-labeled capture antibody is prepared under the premise of many failures:
Effectively by quantum dot and antibody coupling and to keep its biological activity be a vital step, quantum dot and anti-
The ultimate challenge that body is successfully coupled is the stability of coupled product.Theoretically quantum dot is feasible with antibody coupling, but
It is that in real work, quantum dot and antibody coupling are not an easy things, and the present invention is to pay very big labour generation
Valence also has failed many times.The reason of it fails has the following aspects:
1) in step S3), when the pH value of solution is between 5~7, quantum dot agglomerates, and cannot carry out with antibody
Effective coupling；
2) in step S3), if being unsatisfactory for the following conditions: the quantum dot of activation and the ethyl -3- carbonization two being added twice
The mass ratio of imide hydrochloride compound is 5~10, the matter of the quantum dot of activation and the N- hydroxy thiosuccinimide being added twice
When amount is than for 5~10, the carboxyl of quantum dot is slowly combined with the amino of antibody, but combines insecure, occurs having separation again
The phenomenon that；
3) other than present invention setting quantum dot and coupling agent ratio, suitable pH value, the conditions such as BSA, methanol being added,
Inventor is before quantum dot and antibody coupling, using monophosphate sugar to capture antibody modification to get the capture antibody of activation；For
Quantum dot success coupled antibody and change the surface texture of antibody, this is to create for quantum dot with the successful coupling for capturing antibody
Optimum condition.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Claims (8)
1. a kind of for detecting the test card of tumor M 2-type pyruvate kinase, which is characterized in that the test card includes bottom plate, bottom
Plate is equipped with the sequentially connected sample pad in end, bonding pad, nitrocellulose membrane and water absorption pad, the bonding pad along sample flow direction
It is adsorbed with quantum dot-labeled capture antibody, nitrocellulose membrane is successively arranged detection band and quality control band, institute along sample flow direction
It states detection band and is coated with detection antibody, the quality control band is coated with IgG；
The capture antibody is antitumor M2 type pyruvate kinase antibody, and the capture antibody is before marking quantum dot, using list
The surface of the sugar-modified capture antibody of phosphoric acid；The detection antibody is antitumor M2 type pyruvate kinase antibody, the capture antibody
The different epitopes of tumor M 2-type pyruvate kinase in conjunction with detection antibody；
The sugar-modified capture antibody of the monophosphate is to be prepared by the following method: monophosphate sugar being added into capture antibody
With bovine serum albumin(BSA) buffer, 1~2h is placed at room temperature for after shaking up；The pH value of solution is adjusted to 8~11 again.
2. the preparation method of test card as described in claim 1, which comprises the following steps: the bonding pad presses 1
~3 μ L/cm spray the quantum dot-labeled capture antibody-solutions of 0.1~5mg/mL, and the detection band is by 1~3 μ L/cm spraying 0.1
~5mg/mL detects antibody-solutions, and the quality control band sprays 0.1~5mg/mL IgG solution by 1~3 μ L/cm；It is quantum dot-labeled
Capture antibody-solutions, detection antibody-solutions and sheep anti-mouse igg solution solvent be coating buffer, the coating buffer is
Include 20~150mM NaCl, 0.05%~3%PEG (w/v), 0.2%~1% trehalose (w/v), 2~10mg/mL BSA
(w/v), 0.05%NaN310~100mM PBS buffer solution.
3. the preparation method of the test card as described in right will go 2, which is characterized in that the quantum dot-labeled capture antibody is
It is prepared by following steps:
S1) quantum point activation: using N- hydroxy thiosuccinimide by the activated carboxylic of CdSe/ZnS quantum dot surface；
S2) using the surface of the sugar-modified capture antibody of monophosphate to get the capture antibody of activation；
S3) quantum dot of activation is placed to room temperature, ethyl -3- carbodiimides hydrochloride is added into the quantum dot of activation
Solution, N- hydroxy thiosuccinimide solution, BSA solution, are added the capture antibody of the activation after shaking up, carry out after shaking up
It is incubated for, ethyl -3- carbodiimides hydrochloride solution, N- hydroxy thiosuccinimide solution is added during being incubated for；It incubates
Methanol is added after educating, and oscillation is protected from light after mixing；It adds beta -mercaptoethanol to be terminated, uses β-sulfydryl after terminating reaction
Ethyl alcohol is dialysed；Centrifugation removal supernatant is after dialysis to get quantum dot-labeled capture antibody.
4. the preparation method of test card as claimed in claim 3, which is characterized in that in step sl, the quantum dot of activation
Concentration is 1~10 μM；In step s 2, the concentration of the capture antibody is 10~100 μ g/ml；In step s3, the BSA
The concentration of solution is 20~200mg/mL, and the mass ratio of the capture antibody of the quantum dot and activation of activation is (1:2)~(1:10).
5. the preparation method of test card as claimed in claim 2, which is characterized in that the bonding pad is by 2 μ L/cm spraying 0.1
~5mg/mL quantum dot-labeled capture antibody-solutions, the detection band are molten by 2 μ L/cm spraying 0.1~5mg/mL detection antibody
Liquid, the quality control band spray 0.1~5mg/mL IgG solution by 2 μ L/cm.
6. the preparation method of test card as claimed in claim 2, which is characterized in that the sample pad is carried out using pretreatment fluid
It impregnates, then dries；The bonding pad is impregnated before spraying using pretreatment fluid, then is dried；The pretreatment fluid be containing
The 0.01M and pH of 0.02%~0.1%Tween-20 (w/v) is 7~7.3PBS buffer.
7. a kind of kit comprising test card as described in claim 1, the kit also include to get stuck, get stuck including back
Card and upper cover, back card are equipped with card slot, and the test card is embedded in the card slot, and the upper cover is equipped with testing window and well,
The position of the testing window is matched with the position of the detection band and quality control band, the position of the well and the sample pad
Position match.
8. kit as claimed in claim 7, which is characterized in that the kit also includes sample dissociating buffer, described
Sample dissociating buffer be include medium, 0.45%~0.9% sodium chloride (w/v), 0.02%~0.1%Tween-20 (w/
V), the PBS buffer solution of 20~200mg/mL bovine serum albumin(BSA)；When the medium is cesium chloride, in sample dissociating buffer
Cesium chloride concentrations are 10%~30% (v/v)；When the medium is sucrose, sucrose concentration is 0.1 in sample dissociating buffer
~0.5mol/L；When the medium is saccharoSan, saccharoSan concentration is 10%~30% (w/ in sample dissociating buffer
v)。
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Cited By (1)
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