CN109813893A

CN109813893A - CEA test strip and preparation method thereof, CEA detection device

Info

Publication number: CN109813893A
Application number: CN201910081524.0A
Authority: CN
Inventors: 胡鹍辉; 钱纯亘; 吴力强; 张赛; 谢妮; 张德玺
Original assignee: Institute of Biophysics of CAS; Shenzhen Yhlo Biotech Co Ltd; Shenzhen Second Peoples Hospital
Current assignee: Institute of Biophysics of CAS; Shenzhen Yhlo Biotech Co Ltd; Shenzhen Second Peoples Hospital
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2019-05-28

Abstract

The present invention relates to a kind of CEA test strips and preparation method thereof, CEA detection device.The CEA test strip includes bottom plate and loading pad, bonding pad, cushion, coated film and absorption pad set on the bottom plate, loading pad, bonding pad, cushion, coated film and absorption pad are sequentially connected with from one end of bottom plate to the other end, the first CEA monoclonal antibody of nano enzyme label is provided on bonding pad, coated film is equipped with detection line, detection line is provided with the 2nd CEA monoclonal antibody, and the first CEA monoclonal antibody and the 2nd CEA monoclonal antibody of nano enzyme label can be in conjunction with the different epitopes of CEA.The accuracy of above-mentioned CEA test strip is higher.

Description

CEA test strip and preparation method thereof, CEA detection device

Technical field

The present invention relates to medical sciences, examine more particularly to a kind of CEA test strip and preparation method thereof, CEA Survey device.

Background technique

Carcinomebryonic antigen (carcino-embryonic antigen, CEA) is a kind of acid with human embryos antigenic characteristic Property glycoprotein, the immune response of patient can be caused as antigen.Carcinomebryonic antigen can be widely present in the Digestive of endoderm origin System cancer, exists in the digestion tubing of normal fetus, can also there is micro presence in normal human serum.Carcinomebryonic antigen is one A broad spectrum activity tumor markers, are able to reflect out the presence of kinds of tumors, the curative effect of colorectal cancer, breast cancer and lung cancer is judged, Progression of the disease, monitoring and prognosis estimation are a preferable tumor markers.Carcinomebryonic antigen may result from breast tissue, as swollen Tumor markers, which are present in cancerous tissue, to be had been found.There is the content of CEA in studies have shown that nipple discharge in malignant change group, good There were significant differences between venereal disease change group and healthy control group.The measurement of CEA is to the property of clear mammary gland disease in nipple discharge, Auxiliary diagnosis breast cancer is of great significance.

Some researches show that the acquisition of nipple discharge is a kind of safe and reliable, hurtless measure, test side without side-effects for foreign countries Method.In recent years, find nipple discharge in tumor marker, such as carcinomebryonic antigen, prostate specific antigen with it is breast cancer related. In all mammary gland diseases through surgical intervention, nipple discharge is the most common symptom after lump in breast.Most patients The cause of nipple discharge is the benign lesion of mammary gland.According to clinical characteristics, nipple discharge is divided into pathologic and two kinds of physiological.Often The simple physiological discharge seen, it is not related with malignant change, without being handled, such as latter half of gestation or nursing period Nipple discharge.Pathologic discharge may be related with malignant change.

Breast cancer is the highest malignant tumour of developed country's women disease incidence, and the metropolitan breast cancer incidence in China is also in Rise year by year trend.Traditional breast cancer screening method is mainly the CEA content etc. of mammary X-ray photography or measurement serum, but X is penetrated Line also can not be ignored the irreversible damaging action that human body generates.Traditional CEA detection method mainly has enzyme linked immunosorbent assay And chemoluminescence method etc., it is detected using in vitro sample, safety is higher.However, enzyme linked immunosorbent assay and chemiluminescence The marker that method uses is native enzyme, such as horseradish peroxidase (HRP), alkaline phosphatase (AP) mostly.Due to most of Native enzyme is all protein, encounters the non-physiological conditions such as heat, acid, alkali, is easy to happen structure change and loses catalytic activity.

Nano enzyme is the special performance of a kind of existing nano material, and has the analogue enztme of catalysis.Nano enzyme, which has, urges Change that high-efficient, enzymatic activity is stable, economy and the characteristics of prepare with scale, CEA can be detected by nanometer enzymic-labelled antibody, with Overcome the problems, such as that the stability of native enzyme is poor.However during traditional use nanometer enzymic-labelled antibody detects CEA, false yin Property phenomenon than more serious, cause testing result inaccurate.

Summary of the invention

Based on this, it is necessary to provide a kind of higher CEA test strip of accuracy.

In addition, also providing the preparation method and CEA detection device of a kind of CEA test strip.

A kind of CEA test strip, comprising: bottom plate and loading pad on the bottom plate, bonding pad, cushion, Coated film and absorption pad, the loading pad, the bonding pad, the cushion, the coated film and the absorption pad from One end of the bottom plate to the other end is sequentially connected with, and the bonding pad is equipped with the first CEA monoclonal antibody of nano enzyme label, The coated film is equipped with detection line, and the detection line is equipped with the 2nd CEA monoclonal antibody, and the first of nano enzyme label CEA monoclonal antibody and the 2nd CEA monoclonal antibody can be in conjunction with the different epitopes of CEA.

It finds after study, more difficult in conjunction with the CEA also, traditional Test paper of the CEA antibody of nano enzyme label The reaction time of the loading fast speed of item, CEA in sample to be tested and the CEA antibody that nano enzyme marks is shorter, causes to be measured CEA in sample fails to be detected in conjunction with the CEA antibody of nano enzyme label in time, is easy to appear false negative phenomenon.This Mainly loading pad and cushion is respectively set by the two sides in bonding pad in research, and the CEA in sample to be tested and nano enzyme mark The first CEA monoclonal antibody can in the enterprising single step reaction of cushion, extend CEA and nano enzyme label the first CEA it is mono- The reaction time of clonal antibody avoids appearance so that the first CEA monoclonal antibody of nano enzyme label is sufficiently combined with CEA The accuracy of false negative, testing result is higher；Also, it can also make sample to be tested before entering coated film by the way that cushion is arranged What can be distributed is more uniform, can more fully be combined with the 2nd CEA antibody in detection line, further increases detection Accuracy.In addition, by setting can in conjunction with different CEA epitopes make nano enzyme mark the first CEA monoclonal antibody with 2nd CEA monoclonal antibody improves the accuracy of detection so that the combination effect of CEA is more preferable.

The nano enzyme can be catalyzed the colour developing of 3,3', 5,5'- tetramethyl benzidines in one of the embodiments,.

The nano enzyme is magnetic ferroferric oxide nanometer particle, the nano enzyme surface in one of the embodiments, With conjugated group, the conjugated group can be in conjunction with the first CEA monoclonal antibody.

The Avidin of nano enzyme label is additionally provided on the bonding pad in one of the embodiments, on the coated film It is additionally provided with the nature controlling line with the detection line interval, the nature controlling line is located at the detection line close to the side of the absorption pad, The nature controlling line is provided with Avidin antibody.

A kind of preparation method of CEA test strip, includes the following steps:

The first CEA monoclonal antibody that nano enzyme marks is set on polyester cellulose film, it is dry, obtain bonding pad；

2nd CEA monoclonal antibody is set on nitrocellulose filter, it is dry, the coated film with detection line is obtained, The 2nd CEA monoclonal antibody can combine different CEA tables from the first CEA monoclonal antibody that the nano enzyme marks Position；And

The loading pad, bonding pad, cushion, described is sequentially connected with from one end of the bottom plate to the other end on bottom plate Coated film and absorption pad obtain CEA test strip.

The first CEA monoclonal antibody by nano enzyme label is set to polyester fiber in one of the embodiments, Before on plain film, further include the steps that the first CEA monoclonal antibody for preparing the nano enzyme label:

Nano enzyme, carbodiimide and n-hydroxysuccinimide are mixed and reacted, is separated by solid-liquid separation, precipitating is collected；By institute It states precipitating to be reacted with the first CEA monoclonal antibody, obtains the first CEA monoclonal antibody of the nano enzyme label, it is described The mass ratio of first CEA monoclonal antibody and the nano enzyme is 0.1:1~1:1；

Alternatively, mixing and reacting nano enzyme with the first CEA monoclonal antibody, the first of the nano enzyme label is obtained The mass ratio of CEA monoclonal antibody, the first CEA monoclonal antibody and the nano enzyme is 0.2:1~2:1.

It is described in one of the embodiments, to be added into the precipitating after the first CEA monoclonal antibody reacted, Further include following steps: confining liquid is added in the reactant obtained to the precipitating with the first CEA monoclonal antibody reactive It is reacted, the confining liquid includes the PBS buffer solution of 0.01M~0.1M, the ox blood that mass percentage is 0.1%~1% The Triton X-100 that pure albumen and mass percentage are 0.01%~0.1%；

Alternatively, including the following steps: after the nano enzyme is mixed and reacted with the first CEA monoclonal antibody to described Confining liquid is added in the reactant that nano enzyme is obtained with the first CEA monoclonal antibody reactive to be reacted, the confining liquid includes The bovine serum albumin bletilla mass percentage that the PBS buffer solution of 0.01M~0.1M, mass percentage are 0.1%~1% is 0.01%~0.1% Triton X-100.

The first CEA monoclonal antibody is secreted to obtain by hybridoma in one of the embodiments, the hybridization The preparation step of oncocyte are as follows: merged using spleen cell of the CEA after immune with myeloma cell, it is thin to obtain the hybridoma Born of the same parents.

The first CEA monoclonal antibody by nano enzyme label is set to polyester fiber in one of the embodiments, Step on plain film specifically: the first CEA monoclonal antibody for marking the nano enzyme is mixed with treatment fluid, obtains antibody spray Masking liquid；The antibody spray coating liquor is sprayed on the polyester cellulose film；The treatment fluid includes that mass percentage is Trehalose that 0.1%~1% bovine serum albumin(BSA), mass percentage are 5%~20%, mass percentage 0.1% PH7.4~9.0 of~2% casein, the S9 surfactant that mass percentage is 0.1%~1%, 0.01M~0.2M Tris buffer；

And/or described 2nd CEA monoclonal antibody is set to the step on nitrocellulose filter specifically: using packet After the concentration to 0.5mg/mL~2.0mg/mL for being adjusted the 2nd CEA monoclonal antibody by liquid, then draw the cellulose nitrate On plain film, the coating buffer include the PBS buffer solution of 0.01M~0.05M, mass percentage be 5%~20% trehalose, The methanol that mass percentage is 1%~3%.

A kind of CEA detection device, which is characterized in that including above-mentioned CEA test strip or above-mentioned CEA test strip The CEA test strip that is prepared of preparation method.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of the CEA test strip of an embodiment.

Specific embodiment

To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing Give preferred embodiment of the invention.But the invention can be realized in many different forms, however it is not limited to herein Described embodiment.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more saturating It is thorough comprehensive.

Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.

As shown in Figure 1, the CEA test strip 10 of an embodiment can accurately detect the CEA content in sample to be tested, It can be applied in CEA detection device.Sample to be tested is nipple discharge or blood in one of the embodiments,.

Specifically, CEA test strip 10 includes bottom plate 100 and loading pad 200, bonding pad on bottom plate 100 300, cushion 400, coated film 500 and absorption pad 600.Loading pad 200, bonding pad 300, cushion 400, coated film 500 And absorption pad 600 is sequentially connected with from one end of bottom plate 100 to the other end.Bonding pad 300 is equipped with the first of nano enzyme label CEA monoclonal antibody.Coated film 500 is equipped with detection line 510.Detection line 510 is equipped with the 2nd CEA monoclonal antibody.Nano enzyme The first CEA monoclonal antibody and the 2nd CEA monoclonal antibody of label can be in conjunction with the different epitopes of CEA.

By the way that loading pad 200 and cushion 400 is respectively set in the two sides of bonding pad 300, the CEA in sample to be tested with receive First CEA monoclonal antibody of rice enzyme label can extend CEA and nano enzyme marks in the enterprising single step reaction of cushion 400 The first CEA monoclonal antibody reaction time so that nano enzyme label the first CEA monoclonal antibody sufficiently combined with CEA, It avoids and false negative occurs, the accuracy of testing result is higher；Also, sample to be tested can also be made by the way that cushion 400 is arranged Can be distributed before entering coated film 500 it is more uniform, with can be with the 2nd CEA antibody in detection line 510 more fully In conjunction with further increasing the accuracy of detection.In addition, nano enzyme can be made to mark in conjunction with different CEA epitopes by setting The first CEA monoclonal antibody and the 2nd CEA monoclonal antibody, CEA in sample can respectively with the first CEA monoclonal antibody It is combined with the 2nd CEA monoclonal antibody and forms double-antibody sandwich compound, it is more preferable in conjunction with effect, improve the accuracy of detection.

Bottom plate 100 is the supporting body of CEA test strip 10.Bottom plate 100 is that PVC board is (poly- in one of the embodiments, Vinyl chloride bottom plate) or glass substrate etc..In the illustrated embodiment, bottom plate 100 is PVC board.PVC board is frivolous, and has good Corrosion resistance and good waterproof performance avoid antibody and sample leakage to be detected.

Loading pad 200 is polyester cellulose film in one of the embodiments,.Bonding pad 300 is polyester cellulose film.It is slow Rushing pad 400 is polyester cellulose film.Coated film 500 is nitrocellulose filter.Further, coated film 500 is Mi Libo HF120 Nitrocellulose filter.

In one of the embodiments, in the first CEA monoclonal antibody of nano enzyme label, nano enzyme and the first CEA are mono- The mass ratio of clonal antibody is 0.1~1.The response curve that can guarantee under the ratio is more smooth, avoids because antibody is dense Spend it is low cause low side sensitivity lower, also avoid causing high-end gradient insufficient because antibody concentration is excessively high.

Nano enzyme can be catalyzed the colour developing of 3,3', 5,5'- tetramethyl benzidines in one of the embodiments,.3,3',5, 5'- tetramethyl benzidine, that is, TMB is a kind of chromogen reagent of new type of safe.

Nano enzyme is magnetic ferroferric oxide nanometer particle in one of the embodiments,.Further, high molecular polymerization Object is polyvinyl alcohol or polystyrene.

Further, nano enzyme surface has conjugated group, and conjugated group can be in conjunction with the first CEA monoclonal antibody. Specifically, conjugated group, which is selected from, carboxyl (- COOH), amino (- NH₂), tosyl (- CH₃-C₆H₅-SO₂) and chloromethane phenyl (-C₆H₄-CH₂At least one of Cl).

The partial size of nano enzyme is 90nm~200nm in one of the embodiments,.

Nano enzyme is prepared using method disclosed in patent 201610416819.5 in one of the embodiments, Nano enzyme.

The first CEA monoclonal antibody is secreted to obtain by hybridoma in one of the embodiments,.Further, miscellaneous Hand over the preparation step of oncocyte are as follows: merge using spleen cell of the CEA after immune with myeloma cell, obtain hybridoma.

Further, the spleen cell after CEA is immune is the Mouse spleen cells after CEA immune.Myeloma cell is Sp2/0-Ag14 murine myeloma cell.

Specifically, the step of being merged with myeloma cell using spleen cell of the CEA after immune, obtained fused cell is specific Are as follows: by spleen cell of the CEA after immune and myeloma cell with number ratio for (5~10): 1 mix, adds PEG fusion agent solution, After standing 80s~100s, fusion is terminated, positive fused cell is screened with CEA, obtains hybridoma.Wherein, PEG fusion agent The ratio of solution and splenocyte is 1mL:10⁸It is a.

It should be noted that the first CEA monoclonal antibody is not limited by above-mentioned hybridoma secretion, city can be passed through Purchase is sold, such as can be Hytetst company and clone's number CEA monoclonal antibody for being 3C1.

The first CEA monoclonal antibody that nano enzyme marks in one of the embodiments, is made by the steps To: nano enzyme, carbodiimide and n-hydroxysuccinimide are mixed and reacted, is separated by solid-liquid separation, precipitating is collected；It will precipitating and the One CEA monoclonal antibody is reacted, and the first CEA monoclonal antibody of nano enzyme label is obtained.First CEA monoclonal antibody with The mass ratio of nano enzyme is 0.1~1.

Specifically, 10mM~100mM, the MES buffer of pH6.0~8.0 and nano enzyme are mixed, obtains nano enzyme mixing Liquid, the concentration of nano enzyme is 0.2mg/mL~2mg/mL in nanometer enzyme mixation；Final concentration of 0.1mg/mL~5mg/mL is added Carbodiimide and final concentration of 0.1mg/mL~5mg/mL n-hydroxysuccinimide, mix, incubation at room temperature 20min~ 40min is stirred so that nano enzyme is in suspended state during being incubated for；It is separated by solid-liquid separation, collects precipitating；Using 10mM~ The MES buffer of 100mM, pH6.0~8.0 will precipitating redissolve after, be added the first CEA monoclonal antibody react at room temperature 2h~ 4h is separated by solid-liquid separation, discards supernatant, and obtains the first CEA monoclonal antibody of nano enzyme label.First CEA monoclonal antibody with receive The mass ratio of rice enzyme is 0.1~1.

It further, further include adopting before 10mM~100mM, the MES buffer of pH6.0~8.0 and nano enzyme being mixed Nano enzyme is cleaned 1 time~2 times with the MES buffer of 10mM~100mM, pH6.0~8.0.

After precipitating being redissolved using the MES buffer of 10mM~100mM, pH6.0~8.0, it is anti-to be added the first CEA monoclonal Further include following steps before body reacts the step of 2h~4h at room temperature: 10mM~100mM, pH6.0~8.0 MES buffering Liquid cleans precipitating at least 3 times.

After precipitating being redissolved using the MES buffer of 10mM~100mM, pH6.0~8.0, it is anti-to be added the first CEA monoclonal Further include following steps after the step of body is reacted: cleaning precipitating is reacted with what the first CEA monoclonal antibody reactive obtained Object, cleaning solution are the MES buffer of 10mM~100mM, pH6.0~8.0.

Be added in one of the embodiments, into precipitating after the first CEA monoclonal antibody reacted, further include as Lower step: confining liquid is added in the reactant obtained to precipitating with the first CEA monoclonal antibody reactive and is reacted.Confining liquid packet Include the PBS buffer solution of 0.01M~0.1M, the bovine serum albumin bletilla mass percentage that mass percentage is 0.1%~1% For 0.01%~0.1% Triton X-100.The group site being not associated on nano enzyme can be closed using this confining liquid, with Reduce the nonspecific reaction of the first CEA monoclonal antibody of nano enzyme label.Further, be added confining liquid after reaction when Between be at least 1h.

Further, confining liquid is added in the reactant obtained to precipitating with the first CEA monoclonal antibody reactive to carry out instead The step of answering specifically: the reactant that precipitating is obtained with the first CEA monoclonal antibody reactive is separated by solid-liquid separation, it is heavy to obtain reaction It forms sediment；Confining liquid is added into reaction precipitating to be reacted.

Further, confining liquid is added in the reactant obtained to precipitating with the first CEA monoclonal antibody reactive to carry out instead It further include the first CEA monoclonal antibody using confining liquid cleaning nano enzyme label after the step of answering.Wash number is 1 time ~2 times.Further, it is added in the first CEA monoclonal antibody marked to nano enzyme and saves liquid, obtained final concentration of The antibody stock solution of 0.1mg/mL~1mg/mL.Saving liquid includes that the PBS buffer solution of 0.01M~0.1M, mass percentage are 0.1%~1% bovine serum albumin(BSA), the Triton X-100 and quality percentage that mass percentage is 0.01%~0.1% The trehalose that content is 1%~5%.

The Avidin of nano enzyme label is additionally provided on bonding pad 300 in one of the embodiments,.On coated film 500 It is additionally provided with the nature controlling line 520 being spaced with detection line 510.Nature controlling line 520 is located at detection line 510 close to the side of absorption pad 600.Matter It controls line 520 and is equipped with Avidin antibody.

In one of the embodiments, in the Avidin of nano enzyme label, the mass ratio of nano enzyme and Avidin is 0.1~ 1.Further, the preparation of the preparation process of the Avidin of nano enzyme label and the first CEA monoclonal antibody of nano enzyme label Process is roughly the same, the difference is that, the first CEA monoclonal antibody is substituted with Avidin.

Further, detection line 510 crosses over coated film 500 from first direction.First direction be can with cushion 400 to The direction of the line intersection of absorption pad 600.Nature controlling line 520 crosses over coated film 500 from second direction.Second direction be can with it is slow The direction that the line for rushing pad 400 to absorption pad 600 intersects.Such as detection line 510 and nature controlling line 520 across or be inclined cross coated film 500 so that from cushion 400 to absorption pad 600, sample to be detected must line 510 and nature controlling line 520 after testing, prevent There is the result of false negative in missing inspection.

In the illustrated embodiment, first direction is and cushion 400 to the vertical side of the line of line of absorption pad 600 To.Second direction is and cushion 400 to the vertical direction of the line of line of absorption pad 600.Detection line 510 and nature controlling line 520 are arranged in parallel.This design can reduce the dosage of the antibody on detection line 510 and nature controlling line 520, save the cost.

Further, the spacing between detection line 510 and nature controlling line 520 is 4mm~8mm.

In one embodiment, the Avidin antibody on nature controlling line 520 is rabbit-anti Avidin antibody.Rabbit-anti Avidin is anti- Body and Avidin excellent bonding performance, non-specific binding are few.

In one embodiment, absorption pad 600 is the blotting paper with good water absorbing properties, so that sample to be detected Bonding pad 300, cushion 400 and coated film can successively be passed through from loading pad 200s under the attraction of absorption pad 600 500。

In one embodiment, loading pad 200, bonding pad 300, cushion 400, coated film 500 and absorption pad 600 It is successively set on from one end of bottom plate 100 to the other end on bottom plate 100, the length Chong Die with bonding pad 300 of loading pad 200 is 1mm~5mm.The length Chong Die with cushion 400 of bonding pad 300 is 1mm~5mm.Cushion 400 is Chong Die with coated film 500 Length is 1mm~5mm.The length Chong Die with absorption pad 600 of coated film 500 is 1mm~5mm.Under such setting, sample to be detected When this addition loading pad 200, bonding pad 300, cushion 400 and coated film can be successively passed through under the action of absorption pad 600 500, and changed according to the signal value of the detection line of coated film 500 510 and nature controlling line 520, detect containing for CEA in sample to be detected Amount.

Further, loading pad 200, bonding pad 300, cushion 400, coated film 500 and absorption pad 600 are from bottom plate 100 one end to the other end is arranged on bottom plate 100.Bonding pad 300 partly overlaps with loading pad 200.Cushion 400 and combination Pad 300 partly overlaps.Coated film 500 partly overlaps with cushion 400.Absorption pad 600 partly overlaps with coated film 500.

Further, loading pad 200, bonding pad 300, cushion 400, coated film 500 and absorption pad 600 are successively taken It connects.200 part of loading pad is located on side of the bonding pad 300 far from bottom plate 100.It is remote that 300 part of bonding pad is located at cushion 400 On side from bottom plate 100.400 part of cushion is located at side of the coated film 500 far from bottom plate 100.600 part of absorption pad position In on side of the coated film 500 far from bottom plate 100.This design is so that sample to be detected successively passes through from loading pad 200s Bonding pad 300, cushion 400 and coated film 500 flow more smooth.

Specifically, loading pad 200, bonding pad 300, cushion 400, packet are sequentially mutually pasted to overlap joint on bottom plate 100 Envelope 500 and water absorption pad obtain test paper plate, and the test strips of 2.5mm~6mm width are cut into according to cutting requirement, obtain CEA inspection Test paper slip 10.

Loading pad 200, bonding pad 300, cushion 400, coated film 500 and absorption pad in one of the embodiments, 600 length ratio is (10~13): (13~16): (7~10): (25~25): (23~25).Further, loading pad 200, Bonding pad 300, cushion 400, coated film 500 and absorption pad 600 length ratio be 13:13:10:25:23.

The CEA test strip 10 of above embodiment use process is as follows:

In use, sample to be tested is added on the loading pad 200 of above-mentioned CEA test strip 10.In the suction of absorption pad 600 Successively pass through bonding pad 300, cushion 400 and coated film 500 from loading pad 200s under drawing.If containing in test sample CEA (antigen), then CEA is under capillarity in conjunction with the first CEA monoclonal antibody of the nano enzyme label on bonding pad 300 Form compound.The compound is moved to the detection line 510 on coated film 500 under capillarity, and in detection line 510 The 2nd CEA monoclonal antibody reactive and combine, form double-antibody sandwich compound, and be gathered in detection line 510.React 5min After~10min, TMB color developing agent is added, after fully reacting, detection line 510 is in navy blue, and the depth of the color of detection line 510 It is positively correlated with the CEA concentration in sample to be tested.

The Avidin of nano enzyme label on bonding pad 300 is moved to the nature controlling line 520 on coated film 500, with Avidin Antibody response and combine, form nano enzyme-Avidin-antibody complex, and be gathered in nature controlling line 520.React 5min~10min Afterwards, TMB color developing agent is added, after fully reacting, nature controlling line 520 is in navy blue.Under normal circumstances, no matter sample to be detected whether Containing CEA, nature controlling line 520 all should be in navy blue, illustrate that testing result is invalid if nature controlling line 520 is without color change, avoid The testing result of false negative or false positive.

Digital signal is converted by colour difference signal by detection system, using concentration point as abscissa, 510 signal value of detection line It is that ordinate draws standard curve than 520 signal value of nature controlling line (T/C), so as to calculating in sample to be tested for accurate quantitative analysis The concentration of CEA.

The study found that more difficult in conjunction with the CEA also, traditional test strip of CEA antibody of nano enzyme label The reaction time of loading fast speed, CEA in sample to be tested and the CEA antibody that nano enzyme marks is shorter, leads to sample to be tested In CEA fail in time with nano enzyme label CEA antibody in conjunction with and detected, be easy to appear false negative phenomenon.This research Mainly by the way that loading pad 200 and cushion 400 is respectively set in the two sides of bonding pad 300, the CEA and nano enzyme in sample to be tested First CEA monoclonal antibody of label in the enterprising single step reaction of cushion 400, can extend the of CEA and nano enzyme label The reaction time of one CEA monoclonal antibody avoids so that the first CEA monoclonal antibody of nano enzyme label is sufficiently combined with CEA There is false negative, the accuracy of testing result is higher；Also, by be arranged cushion 400 can also make sample to be tested into Enter can be distributed before coated film 500 it is more uniform, can more fully be combined with the 2nd CEA antibody in detection line 510, Further increase the accuracy of detection.In addition, can be in conjunction with make nano enzyme label first of different CEA epitopes by setting CEA monoclonal antibody improves the accuracy of detection so that the combination effect of CEA is more preferable with the 2nd CEA monoclonal antibody.

The marker that traditional colloidal gold method uses is colloidal gold, and colloidal gold is inorganic matter, and property is stablized, but colloidal gold The sensitivity of method is poor, and the accuracy of testing result is lower when sample size is less.This research uses the first CEA of nano enzyme label Monoclonal antibody, and cushion 400 is set, so that detection sensitivity is higher, minimum detection limit contains CEA up to 0.1ng/mL Measuring less sample to be tested also can accurately detect.

Traditionally, breast cancer is determined whether by CEA content in detection serum.CEA may result from tumor of breast group It knits, is secreted into breast duct first by the CEA that epithelial cell generates, so in early-stage breast cancer and precancerous lesion patients serum In CEA value be not obvious raising, therefore the raising of change of serum C EA value is mainly seen in the advanced cancer case for having transfer, is mainly used for The judgement of breast cancer patients with terminal prognosis, change of serum C EA detection is difficult to diagnosis to early-stage breast cancer and screening provides help.It is above-mentioned The CEA test strips of embodiment can not only detect the CEA content in blood, additionally it is possible to the CEA content in nipple discharge is detected, So as to carry out diagnosis and screening to early-stage breast cancer.

In above-mentioned CEA test strip 10, detection line 510 and nature controlling line 520 are spaced, and the color change of detection line 510 takes The luminous intensity of CEA content certainly in sample, nature controlling line 520 depends on Avidin, and the two does not interfere with each other and influences.It can adopt It is quantified with the mode of T/C value, ensure that the accuracy of test result.And upper test strips are in use, easy to operate, cost It is low.The detector used is not necessarily to professional operator, testing result can be obtained within 15 minutes.

It is appreciated that the first CEA monoclonal antibody of nano enzyme label is not limited by above-mentioned steps preparation, in other realities It applies in example, the first CEA monoclonal antibody of nano enzyme label can be made by the steps to obtain: by nano enzyme and first CEA monoclonal antibody is mixed and is reacted, and obtains the first CEA monoclonal antibody of nano enzyme label.First CEA monoclonal antibody with The mass ratio of nano enzyme is 0.2~2.

Specifically, 10mM~100mM, the MES buffer of pH6.0~8.0 and nano enzyme are mixed, obtains nano enzyme mixing Liquid, the concentration of nano enzyme is 1mg/mL~10mg/mL in nanometer enzyme mixation；By 10mM~100mM, the MES of pH6.0~8.0 Buffer and the first CEA monoclonal antibody mix, and obtain antibody mixed liquor；Nanometer enzyme mixation is mixed with antibody mixed liquor, 2h~4h is reacted at room temperature, obtains the first CEA monoclonal antibody of nano enzyme label.First CEA monoclonal antibody and nanometer The mass ratio of enzyme is 0.2:1~2:1.

It further include as follows after nano enzyme is mixed and reacted with the first CEA monoclonal antibody in one of the embodiments, Step: confining liquid is added in the reactant obtained to nano enzyme with the first CEA monoclonal antibody reactive and is reacted.Confining liquid packet Include the PBS buffer solution of 0.01M~0.1M, the bovine serum albumin bletilla mass percentage that mass percentage is 0.1%~1% For 0.01%~0.1% Triton X-100.The group site being not associated on nano enzyme can be closed using this confining liquid, with Reduce the nonspecific reaction of the first CEA monoclonal antibody of nano enzyme label.Further, be added confining liquid after reaction when Between be at least 1h.

Further, confining liquid is added in the reactant obtained to nano enzyme and the first CEA monoclonal antibody reactive to carry out It further include the first CEA monoclonal antibody using confining liquid cleaning nano enzyme label after the step of reaction.Wash number is 1 It is secondary~2 times.Further, it is added in the first CEA monoclonal antibody marked to nano enzyme and saves liquid, obtained final concentration of The antibody stock solution of 0.1mg/mL~1mg/mL.Saving liquid includes that the PBS buffer solution of 0.01M~0.1M, mass percentage are 0.1%~1% bovine serum albumin(BSA), the Triton X-100 and quality percentage that mass percentage is 0.01%~0.1% The trehalose that content is 1%~5%.

This research also provides the preparation method of the CEA test strip of an embodiment, include the following steps S110~ S130:

S110, the first CEA monoclonal antibody that nano enzyme marks is set on polyester cellulose film, it is dry, it is tied Close pad.

S110 in one of the embodiments, specifically: by the first CEA monoclonal antibody and nano enzyme of nano enzyme label The Avidin of label is respectively arranged to polyester cellulose film, dry, obtains bonding pad.

Specifically, the step the first CEA monoclonal antibody that nano enzyme marks being set on polyester cellulose film is specific Are as follows: the first CEA monoclonal antibody that nano enzyme marks is mixed with treatment fluid, obtains antibody spray coating liquor；Antibody spray coating liquor is sprayed It is applied on polyester cellulose film.Treatment fluid includes the bovine serum albumin(BSA) that mass percentage is 0.1%~1%, quality percentage Casein that trehalose that content is 5%~20%, mass percentage are 0.1%~2%, mass percentage 0.1% The Tris buffer of pH7.4~9.0 of~1% S9 surfactant, 0.01M~0.2M.Further, nano enzyme is marked Avidin be set to the step on polyester cellulose film specifically: by nano enzyme mark Avidin mixed with treatment fluid, Obtain Avidin spray coating liquor；Avidin spray coating liquor is sprayed on polyester cellulose film.

Antibody spray coating liquor is made using the first CEA monoclonal antibody that above-mentioned treatment fluid marks nano enzyme, so that nanometer First CEA monoclonal antibody of enzyme label can adhere better on polyester cellulose film, avoid because of nano enzyme label First CEA monoclonal antibody is detached from and the problem of false negative occurs.It should be noted that the first CEA of above-mentioned nano enzyme label The mode that the Avidin of monoclonal antibody and nano enzyme label is not limited by spraying is set on polyester fiber film, can also be led to The mode for crossing coating is set on polyester fiber film.

Further, the mass percentage for the first CEA monoclonal antibody that nano enzyme marks in antibody spray coating liquor is 5% ~25%.The discharge rate of antibody spray coating liquor is 4 μ of μ L/cm~8 L/cm.It is such that the sensitivity that can be improved CEA test strip is set And accuracy.The mass percentage for the Avidin that nano enzyme marks in Avidin spray coating liquor is 5%~25%.Avidin spraying The discharge rate of liquid is 4 μ of μ L/cm~8 L/cm.

The step of polyester cellulose film is dried in one of the embodiments, specifically: by polyester cellulose film As for humidity less than 15%, 25 DEG C~50 DEG C at dry 12h~for 24 hours, obtain bonding pad.

Nano enzyme can be catalyzed the colour developing of 3,3', 5,5'- tetramethyl benzidines in one of the embodiments,.

Nano enzyme is that magnetic ferroferric oxide magnetic Nano enzyme or high molecular polymer are received in one of the embodiments, Rice enzyme.Nano enzyme surface has conjugated group, and conjugated group can be in conjunction with the first CEA monoclonal antibody.Further, in conjunction with Group, which is selected from, carboxyl (- COOH), amino (- NH₂), tosyl (- CH₃-C₆H₅-SO₂) and chloromethane phenyl (- C₆H₄- CH₂At least one of Cl).

The partial size of nano enzyme is 90nm~200nm in one of the embodiments,.

The first CEA monoclonal antibody is secreted to obtain by hybridoma in one of the embodiments,.Further, The preparation step of one CEA monoclonal antibody includes S111~S113:

S111, it is merged using spleen cell of the CEA after immune with myeloma cell, obtains fused cell.

Specifically, the step of being merged with myeloma cell using spleen cell of the CEA after immune, obtained fused cell is specific Are as follows: by spleen cell of the CEA after immune and myeloma cell with number ratio for (5~10): 1 mix, adds PEG fusion agent solution, After standing 90s, fusion is terminated, fused cell is obtained.Wherein, it is 1mL:10 that PEG, which merges agent solution and the ratio of splenocyte,⁸It is a.

Further, the spleen cell after CEA is immune is the spleen cell of CEA immunized mice.Myeloma cell is Sp2/0-Ag14 murine myeloma cell.

The preparation process of spleen cell after CEA is immune in one of the embodiments, includes S1111~S1114:

S1111, to mouse initial immunity, obtain the mouse of initial immunity.

Specifically, by after the emulsification of CEA antigen, with 90 μ g/, only~110 BALB/c mouse is immunized in the antigen dose of μ g/ only, is raised It supports 1 week, obtains the mouse of initial immunity.Antigen dose refers to the content of CEA.

More specifically, 1:1 is mixed by volume with Freund's complete adjuvant (FCA) by CEA, emulsifier is obtained.By emulsifier With 90 μ g/, only~110 BALB/c mouse is immunized in the antigen dose of μ g/ only, is raised 1 week, is obtained the mouse of initial immunity.Immune side Formula is that subcutaneous (SQ) 5 point mode is injected.

CEA antigen is the CEA antigen that XEMA company article No. is R224 in one of the embodiments,.

S1112, the first booster immunization is carried out to the mouse of initial immunity, obtains the mouse of the first booster immunization.

Specifically, after CEA being emulsified, with 40 μ g/, only~60 the antigen dose of μ g/ only carries out the to the mouse of initial immunity One booster immunization obtains the mouse of the first booster immunization after raising 1 week.

More specifically, the antigen dose after CEA is mixed with incomplete Freund's adjuvant (FIA) with g/~60 μ g/ of 40 μ only First booster immunization is carried out after raising 2 weeks to the mouse of above-mentioned initial immunity and obtains the mouse of the first booster immunization.Reinforcement is exempted from The mode of epidemic disease is immune for leg muscle.

S1113, one or many booster immunizations are carried out to the mouse of first time booster immunization according to the operation of S1112, obtained Mouse is immunized to CEA.The time interval of adjacent booster immunization is 2 weeks.

S1114, the splenocyte for acquiring CEA immunized mice obtain the spleen cell after CEA is immunized, are melted with carrying out cell It closes.

S112, fused cell is screened, positive fused cell is obtained with CEA coated elisa plate using enzyme-linked immunization, and By positive fused cell monoclonal, hybridoma is obtained.

Specifically, culture fused cell is screened to obtain positive fused cell with Dot-ELISA to suitable concentration.It will After positive fused cell culture to suitable cell concentration, by limiting dilution assay by positive fused cell monoclonal, obtain Positive hybridoma cell.

S113, by hybridoma in vitro culture, obtain CEA monoclonal antibody.

Specifically, hybridoma is cultivated into certain time in culture bottle or tank, culture solution is separated by solid-liquid separation, in collection Clearly, supernatant is purified, obtains the first CEA monoclonal antibody.

Wherein, condition of culture is 37 DEG C, 5% carbon dioxide, and incubation time is for 24 hours~48h.Way of purification is affinity chromatography Purifying.Further, way of purification is albumin A or protein g affinity chromatography.Specifically, pass through albumin A or Protein G and CEA Dan Ke Grand antibody, which passes through, to be covalently bonded on the solid phase carrier of activation, and anti-CEA monoclonal antibody is then made by way of changing pH Elution separation.

It should be noted that the first CEA monoclonal antibody is not limited by above-mentioned hybridoma secretion, city can be passed through Purchase is sold, such as can be Hytest company and CEA monoclonal antibody that article No. is 3C1.

It further include the first CEA monoclonal antibody for preparing nano enzyme label in one of the embodiments, before S110 Step: mixing nano enzyme, carbodiimide and n-hydroxysuccinimide and react, is separated by solid-liquid separation, and collects precipitating；Will precipitating with First CEA monoclonal antibody is reacted, and the first CEA monoclonal antibody of nano enzyme label is obtained.First CEA monoclonal antibody Mass ratio with nano enzyme is 0.1~1.

Specifically, 10mM~100mM, the MES buffer of pH6.0~8.0 and nano enzyme are mixed, obtains nano enzyme mixing Liquid, the concentration of nano enzyme is 0.2mg/mL~2mg/mL in nanometer enzyme mixation；Final concentration of 0.1mg/mL~5mg/mL is added Carbodiimide and final concentration of 0.1mg/mL~5mg/mL n-hydroxysuccinimide, mix, incubation at room temperature 20min~ 40min is stirred so that nano enzyme is in suspended state during being incubated for；It is separated by solid-liquid separation, collects precipitating；Using 10mM~ The MES buffer of 100mM, pH6.0~8.0 will precipitating redissolve after, be added the first CEA monoclonal antibody react at room temperature 2h~ 4h is separated by solid-liquid separation, discards supernatant, and obtains the first CEA monoclonal antibody of nano enzyme label.First CEA monoclonal antibody with receive The mass ratio of rice enzyme is 0.1:1~1:1.

In one of the embodiments, before S110, further include the steps that the Avidin for preparing nano enzyme label.Nano enzyme Mark the preparation process of Avidin roughly the same with the preparation process of the first CEA monoclonal antibody that above-mentioned nano enzyme marks, no It is same to be: the first CEA monoclonal antibody is replaced with Avidin.

S120, the 2nd CEA monoclonal antibody is set on nitrocellulose filter, it is dry, obtain the packet with detection line First CEA monoclonal antibody of envelope, the 2nd CEA monoclonal antibody and nano enzyme label can be in conjunction with the different epitopes of CEA.

Specifically, the 2nd CEA monoclonal antibody is set on nitrocellulose filter, Avidin antibody is set to nitric acid It is dry on tunica fibrosa, coated film is obtained, coated film has spaced detection line and nature controlling line.Wherein, detection line is by the 2nd CEA Monoclonal antibody drying is formed, and nature controlling line is formed by the drying of Avidin antibody.

It is specific that the 2nd CEA monoclonal antibody is set to the step on nitrocellulose filter in one of the embodiments, Are as follows: after the concentration to 0.5mg/mL~2.0mg/mL for adjusting the 2nd CEA monoclonal antibody using coating buffer, then draw cellulose nitrate On plain film.Coating buffer include the PBS buffer solution of 0.01M~0.05M, mass percentage be 5%~20% trehalose, quality The methanol that percentage composition is 1%~3%.Further, step Avidin antibody being set on nitrocellulose membrane specifically: After the concentration to 0.5mg/mL~2.0mg/mL for adjusting Avidin antibody using coating buffer, then draw on nitrocellulose filter.This Kind setting, enable the 2nd CEA monoclonal antibody and Avidin antibody it is stronger be fixed on coated film.Wherein, it adopts It is crossed with spray-filming agent.

Further, dosage of the coating buffer in scratching process containing the 2nd CEA monoclonal antibody be 0.05 μ L/mm~ 0.2μL/mm.Dosage of the coating buffer containing Avidin antibody in scratching process is 0.05 μ of μ L/mm~0.2 L/mm.

The operation that scribed nitrocellulose filter is dried in one of the embodiments, specifically: will cross Nitrocellulose filter afterwards as humidity less than 15%, 25 DEG C~50 DEG C at dry 12h~for 24 hours, obtain coated film.

One embodiment wherein, the preparation of the preparation process and the first CEA monoclonal antibody of the 2nd CEA monoclonal antibody Process is roughly the same, the difference is that: the site for the CEA antigen being directed to is different, and different hybridomas is selected to be prepared.

It should be noted that the 2nd CEA monoclonal antibody is not limited by above-mentioned hybridoma secretion, city can be passed through Purchase is sold, such as can be Hytest company and CEA monoclonal antibody that article No. is 3C6.

S130, from one end of bottom plate to the other end loading pad, bonding pad, cushion, coated film are sequentially connected on bottom plate And absorption pad, bonding pad is Chong Die with loading pad part, and cushion partly overlaps with bonding pad, coated film and bumper portion weight Folded, absorption pad partly overlaps with coated film, obtains CEA test strip.

In one of the embodiments, before S130, further includes the steps that preparing loading pad: polyester cellulose film is used After confining liquid impregnates, being placed in humidity is dry 12h at < 15%, 25 DEG C~50 DEG C~for 24 hours, obtains loading pad.Confining liquid includes Tritonx-100 that Tris, the mass percentage of 0.01M~0.2M is 0.1%~2%, mass percentage be 0.1%~ The PEG 1000 that 1% BSA (i.e. bovine serum albumin(BSA)) and mass percentage is 0.1%~2%.

In one of the embodiments, before S130, further include the steps that preparing cushion.The preparation process of cushion It is identical as the preparation process of loading pad.

In the preparation method of the CEA test strip of above embodiment, by select suitable coating buffer, confining liquid and Treatment fluid, and optimize CEA test strip as a result, it is possible to prepare that testing result accuracy is higher, with higher sensitivity CEA inspection Paper slip is tested, and then can be applied in CEA detection device.

It is appreciated that the first CEA monoclonal antibody of nano enzyme label is not limited by above-mentioned steps preparation, in other realities It applies in example, the first CEA monoclonal antibody of nano enzyme label can be made by the steps to obtain: by nano enzyme and first CEA monoclonal antibody is mixed and is reacted, and obtains the first CEA monoclonal antibody of nano enzyme label.First CEA monoclonal antibody with The mass ratio of nano enzyme is 0.2:1~2:1.

The following are specific embodiment parts.

It in embodiment if not otherwise indicated using drug and instrument, is this field conventional selection.It is not specified in embodiment The experimental method of actual conditions, usually according to normal condition, such as condition described in document, books or kit factory The method that family is recommended is realized.

If not otherwise specified, in following embodiment, CEA is purchased from XEMA company and article No. is R224.Avidin is purchased from Wuhan Three cross company.Avidin antibody is rabbit-anti Avidin antibody, is purchased from Wuhan San Du company.Bottom plate is PVC board.The purchase of 1640 culture mediums In gibco company.Freund's complete adjuvant is purchased from sigma company.Incomplete Freund's adjuvant is purchased from sigma company.

Embodiment 1

The preparation of hybridoma:

(1) after CEA is mixed with Freund's complete adjuvant with volume ratio for 1, by the dose immunization BALB/c of every 100 μ g of mouse Mouse (BALB/c mouse is purchased from company, Guangdong Medical Lab Animal Center) carries out initial immunity.After raising 1 week, obtain first Immune mouse.

(2) CEA and incomplete Freund's adjuvant with volume ratio be 1 mix after, dosage by every 50 μ g of mouse to exempting from for the first time The mouse of epidemic disease carries out booster immunization.After raising 2 weeks, reinforced again with same dosage, obtains CEA and mouse is immunized.

(3) spleen of mouse, the splenocyte after isolated CEA is immune is immunized in acquisition CEA.

(4) splenocyte by CEA after immune and Sp2/0-Ag14 murine myeloma cell are mixed with number ratio for 8:1, are added The PEG4000 of 1mL merges agent solution (adding in 60s), after standing 90s, terminates fusion, and supernatant is abandoned in centrifugation, and precipitating is gently resuspended Cell.Cell suspension addition has been covered in 96 well culture plates of feeder layer.Then culture plate is set 37 DEG C, contains 5% CO₂Incubator in culture, obtain fused cell.

(5) Dot-ELISA is used, using CEA wrapper sheet, detection, screening fused cell detect positive cell hole, lead to Limiting dilution is crossed, the hybridoma that can secrete the first CEA monoclonal antibody is obtained and the 2nd CEA monoclonal can be secreted is anti- The hybridoma of body.

Embodiment 2

The preparation of monoclonal antibody

(1) two kinds of hybridomas of embodiment 1 being placed in 1640 culture mediums and is cultivated, condition of culture is 37 DEG C, 5% carbon dioxide cultivates 36h.Culture solution is collected, 1200rpm is centrifuged 5min, collects supernatant.

(2) supernatant is subjected to albumin A affinity purification, collects eluent, obtains the first CEA monoclonal antibody and second CEA monoclonal antibody.

Embodiment 3

The preparation of first CEA monoclonal antibody of the nano enzyme label of the present embodiment

(1) nano enzyme is cleaned 1 time using the MES buffer of 50mM, pH7.Nano enzyme is receiving for ferroso-ferric oxide package Rice enzyme, there is carboxyl on nano enzyme surface.Nano enzyme is the nanometer being prepared using method disclosed in patent 201610416819.5 Enzyme.

(2) the MES buffer and nano enzyme of 50mM, pH7 are mixed, obtains a nanometer enzyme mixation, in nanometer enzyme mixation The concentration of nano enzyme is 1mg/mL；The carbodiimide of final concentration of 3mg/mL and the N- hydroxyl amber of final concentration of 2.5mg/mL is added Amber acid imide mixes, and is incubated at room temperature 30min, is stirred during being incubated for so that nano enzyme is in suspended state；It is separated by solid-liquid separation, Collect precipitating.

(3) the first CEA monoclonal antibody is added into precipitating and reacts 2h at room temperature, be separated by solid-liquid separation, discard supernatant, collect Precipitating；It is precipitated using the MES buffer solution for cleaning of 10mM~100mM, pH6.0~8.0 three times, using 10mM~100mM, pH6.0 After~8.0 MES buffer redissolves precipitating, confining liquid is added and carries out reaction 1h, is separated by solid-liquid separation, collects reaction precipitating.Closing Liquid include the PBS buffer solution of 0.05M, mass percentage be 0.6% bovine serum albumin bletilla mass percentage be 0.05% Triton X-100.The mass ratio of first CEA monoclonal antibody and nano enzyme is 0.6.It is anti-using confining liquid cleaning It should precipitate 1 time, be separated by solid-liquid separation, collect precipitating, as the first CEA monoclonal antibody of nano enzyme label.

(5) it is added in the first CEA monoclonal antibody marked to nano enzyme and saves liquid, obtain final concentration of 0.6mg/mL's Antibody stock solution.Save the bovine serum albumin(BSA), quality that liquid includes the PBS buffer solution of 0.05M, mass percentage is 0.5% The trehalose that the Triton X-100 and mass percentage that percentage composition is 0.06% are 3%.

First CEA of the nano enzyme label of the preparation process and the present embodiment of the Avidin of the nano enzyme label of the present embodiment The preparation process of monoclonal antibody is identical, the difference is that, the first CEA monoclonal antibody is substituted with Avidin.

The preparation process of the CEA test strip of the present embodiment is as follows:

(1) the first CEA monoclonal antibody that nano enzyme marks is mixed with treatment fluid, obtains antibody spray coating liquor；By antibody Spray coating liquor is sprayed on polyester cellulose film.Treatment fluid includes the bovine serum albumin(BSA) that mass percentage is 0.5%, quality hundred The S9 table that casein that trehalose that point content is 12.5%, mass percentage are 0.6%, mass percentage are 0.55% Face activating agent, 0.1M pH8 Tris buffer.The matter for the first CEA monoclonal antibody that nano enzyme marks in antibody spray coating liquor Measuring percentage composition is 15%.The discharge rate of antibody spray coating liquor is 6 μ L/cm.The Avidin that nano enzyme marks is mixed with above-mentioned treatment fluid It closes, obtains Avidin spray coating liquor；Avidin spray coating liquor is sprayed on polyester cellulose film.Nano enzyme mark in Avidin spray coating liquor The mass percentage of the Avidin of note is 15%.The discharge rate of Avidin spray coating liquor is 6 μ L/cm.By the polyester fiber after spraying Plain film as humidity less than 15%, 50 DEG C at dry 12h, obtain bonding pad.

(2) after the concentration to 1mg/mL for adjusting the 2nd CEA monoclonal antibody using coating buffer, then nitrocellulose filter is drawn On.Dosage of the coating buffer containing the 2nd CEA monoclonal antibody in scratching process is 0.1 μ L/mm.Coating buffer includes 0.03M PBS buffer solution, mass percentage be 13% trehalose, mass percentage be 2% methanol.It is adjusted using coating buffer After the concentration to 10mg/mL of Avidin antibody, then draw on nitrocellulose filter.The dosage of coating buffer containing Avidin antibody For 0.13 μ L/mm.By scribed nitrocellulose filter as humidity less than 15%, 50 DEG C at dry 12h, obtain coated film. Coated film has spaced detection line and nature controlling line.Wherein, detection line is formed by the drying of the 2nd CEA monoclonal antibody, nature controlling line It is formed by the drying of Avidin antibody.

(3) after impregnating polyester cellulose film with confining liquid, being placed in humidity is that drying for 24 hours, obtains at < 15%, 25 DEG C Sample pad.Confining liquid includes Tritonx-100, the mass percentage 0.6% that the Tris of 0.1M, mass percentage are 1% BSA (i.e. bovine serum albumin(BSA)) and mass percentage be 1% PEG1000.

(4) after impregnating polyester cellulose film with confining liquid, being placed in humidity is dry 12h at < 15%, 50 DEG C, is delayed Punching pad.Confining liquid includes Tritonx-100, the mass percentage 0.6% that the Tris of 0.1M, mass percentage are 1% BSA (i.e. bovine serum albumin(BSA)) and mass percentage be 1% PEG1000.

(5) sequentially mutually overlap joint loading pad, bonding pad, cushion, coated film and water absorption pad is pasted on bottom plate to obtain Test paper plate is cut into the test strips of 4mm width according to cutting requirement, obtains CEA test strip.Loading pad part, which is located at, to be combined It pads on the side far from bottom plate, the length of overlapping is 2mm.It is located at cushion far from the side of bottom plate in conjunction with pad part, is overlapped Length be 3mm.Bumper portion is located at side of the coated film far from bottom plate, and the length of overlapping is 2mm.Pad part is absorbed to be located at For coated film far from the side of bottom plate, the length of overlapping is 2mm.Loading pad, bonding pad, cushion, coated film and water absorption pad Length ratio is 13:13:10:25:23.

Embodiment 4

According to the first CEA monoclonal antibody for preparing nano enzyme label of embodiment 3 and the Avidin of nano enzyme label.

(1) according to the operation preparation bonding pad of embodiment 3.

(2) after the concentration to 10mg/mL for adjusting the 2nd CEA monoclonal antibody using coating buffer, then nitrocellulose is drawn On film.Dosage of the coating buffer containing the 2nd CEA monoclonal antibody in scratching process is 0.1 μ L/mm.Coating buffer includes The trehalose that the PB buffer and mass percentage of 0.01M is 5%.Using coating buffer adjust Avidin antibody concentration to After 10mg/mL, then draw on nitrocellulose filter.The dosage of coating buffer containing Avidin antibody is 0.13 μ L/mm.It will scribing line Nitrocellulose filter afterwards as humidity less than 15%, 50 DEG C at dry 12h, obtain coated film.Coated film has spaced inspection Survey line and nature controlling line.Wherein, detection line is formed by the drying of the 2nd CEA monoclonal antibody, and nature controlling line is by the dry shape of Avidin antibody At.

(3) according to the operation preparation loading pad and cushion of embodiment 3.

(4) it according to operation assembling bottom edge, loading pad, bonding pad, cushion, coated film and the water absorption pad of embodiment 3, obtains CEA test strip.

Embodiment 5

The preparation process of first CEA monoclonal antibody of the nano enzyme label of the present embodiment is as follows:

(1) nano enzyme is cleaned 1 time using the MES buffer of 50mM, pH7.0.Nano enzyme is receiving for polystyrene package Rice enzyme, there is toluenesulfonic acid base on nano enzyme surface.Nano enzyme is to be prepared using method disclosed in patent 201610416819.5 Nano enzyme.

(2) the MES buffer and nano enzyme of 50mM, pH7.0 are mixed, obtains a nanometer enzyme mixation, nanometer enzyme mixation The concentration of middle nano enzyme is 5mg/mL；The MES buffer of 50mM, pH7.0 and the first CEA monoclonal antibody are mixed, resisted Body mixed liquor；Nanometer enzyme mixation is mixed with antibody mixed liquor, 3h is reacted at room temperature, obtains reaction solution.First CEA Dan Ke The mass ratio of grand antibody and nano enzyme is 1.1.

(3) confining liquid is added into reactant and carries out reaction 30min, be separated by solid-liquid separation, collect precipitating, as nano enzyme marks The first CEA monoclonal antibody.The ox blood that confining liquid includes the PBS buffer solution of 0.06M, mass percentage is 0.5% is pure The Triton X-100 that albumen and mass percentage are 0.05%.

(4) it is added in the first CEA monoclonal antibody marked to nano enzyme and saves liquid, obtain final concentration of 0.6mg/mL's Antibody stock solution.Save the bovine serum albumin(BSA), quality that liquid includes the PBS buffer solution of 0.05M, mass percentage is 0.5% The trehalose that the Triton X-100 and mass percentage that percentage composition is 0.06% are 3%.

According to the Avidin of the preparation of embodiment 3 and nano enzyme label.

CEA test strip is prepared according to the preparation process of embodiment 3.

Embodiment 6

The preparation process of first CEA monoclonal antibody of the nano enzyme label of the present embodiment is roughly the same with embodiment 3, The difference is that the first CEA monoclonal antibody is purchased from Hytest company and article No. is the CEA monoclonal antibody of 3C1.

The preparation process of the CEA test strip of the present embodiment is roughly the same with embodiment 3, the difference is that, the Two CEA monoclonal antibodies are purchased from Hytest company and article No. is the CEA monoclonal antibody of 3C6.

Embodiment 7

The preparation process of first CEA monoclonal antibody of the nano enzyme label of the present embodiment is roughly the same with embodiment 3, The difference is that confining liquid includes: the PBS buffer solution of 0.01M, the bovine serum albumin bletilla matter that mass percentage is 1% Measure the Triton X-100 that percentage composition is 0.01%.

Embodiment 8

The preparation process of the CEA test strip of the present embodiment is roughly the same with embodiment 3, the difference is that: this reality Applying example does not have a cushion, the present embodiment by sequentially mutually pasted to overlap joint on bottom plate loading pad, bonding pad, coated film and Water absorption pad obtains test paper plate.Loading pad part is located at bonding pad far from the side of bottom plate, and the length of overlapping is 2mm.Bonding pad Part is located at coated film far from the side of bottom plate, and the length of overlapping is 3mm.It absorbs pad part and is located at coated film far from bottom plate On side, the length of overlapping is 2mm.It is cut into the test strips of 4mm width according to cutting requirement, obtains CEA test strip.On Sample pad, bonding pad, coated film and water absorption pad length ratio be 15:13:25:13.

Test:

Test case 1

Measure the content of CEA in sample to be tested 1 and sample to be tested 2 respectively with the CEA test strip of embodiment 3~8, and Using the content (as comparative example) of CEA in the CEA kit measurement sample to be tested of Roche Holding Ag, replication 10 times, ask flat The result of mean value, measurement indicates that see Table 1 for details for measurement result with average value ± variance.The CEA inspection for the embodiment 3~8 that table 1 indicates Test paper slip, Roche Holding Ag CEA detection kit measurement sample to be tested in CEA content.Wherein, sample to be tested 1 be containing There is the blood serum sample of the CEA of 30ng/mL.Sample to be tested 2 is the nipple discharge sample of the CEA containing 200ng/mL.

Specifically, as follows the step of the concentration of CEA in CEA Test paper quantitative detection sample to be tested:

(1) Specification Curve of Increasing

By CEA antigen be configured to 1000ng/mL, 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 0ng/mL test card, with a batch of reagent, each concentration point is tested 10 times.With detection line (T band), nature controlling line (C band) The ratio of signal value be ordinate, CEA reference material concentration is abscissa, establish equation and be fitted to standard curve, will mark it is bent Information is write in ID chip with burn recording software.

(2) detection of sample:

Detector bar is taken out from kit, after tearing packaging of aluminium foil bag, is laid flat detector bar, is balanced 5 minutes.Using dilution Sample to be tested is diluted 100 times, the sample to be tested after then taking 60 μ L to dilute is added in well, reacts at room temperature 10 minutes.Instead After answering, the TMB developing solution of 20 μ L is added into well, reacts 10 minutes.ID chip is inserted into immunity analysis instrument, will be examined It surveys card insertion and enters immunity analysis instrument card inserting mouth, click " test ", instrument calculates CEA in sample to be tested by analyzing software automatically Concentration.

Table 1

As it can be seen from table 1 in the sample to be tested 1 for using the test strips of embodiment 3 to measure CEA concentration for (28.82 ± 2.06) ng/mL is not significantly different with 30ng/mL；Embodiment 3 measure sample to be tested 2 in CEA concentration be (189.21 ± 16.2) ng/mL is not significantly different with 200ng/mL, illustrates that CEA is dense in the sample to be tested 1 of the test strips measurement of embodiment 8 Degree is (31.22 ± 5.08) ng/mL, is not significantly different with 30ng/mL, but whole variance is larger, and accuracy is not good enough；Embodiment CEA concentration in the sample to be tested 2 of 8 measurements is (215.08 ± 27.77) ng/mL, is not significantly different with 200ng/mL, but whole Body variance is larger, and accuracy is not good enough.Illustrate in above embodiment, loading pad is respectively set by the two sides in bonding pad and delays Punching pad, can be improved the accuracy of testing result.

Wherein, compared with Example 3, the CEA concentration for the sample to be tested 1 that embodiment 6 measures is significantly lower than 30ng/mL, to CEA concentration in sample 2 is significantly lower than 200ng/mL, illustrates anti-using the first CEA monoclonal of above embodiment preparation Body and the 2nd CEA monoclonal antibody are more advantageous to the accuracy for improving testing result.Compared with Example 3, embodiment 4 measures The CEA concentration of sample to be tested 1 is significantly lower than 30ng/mL, and the CEA concentration in sample to be tested 2 is significantly lower than 200ng/mL, and explanation is adopted More efficiently the 2nd CEA monoclonal antibody can be coated on coated film with the coating buffer of above embodiment, to protect Demonstrate,prove the accuracy of detection.Compared with Example 3, the CEA concentration for the sample to be tested 1 that embodiment 7 measures is significantly lower than 30ng/mL, CEA concentration in sample to be tested 2 is significantly lower than 200ng/mL, and illustrating can be more effectively using the confining liquid of above embodiment First CEA monoclonal antibody is closed on bonding pad, to guarantee the accuracy of detection.

Test case 2

The nipple discharge sample of 40 patients is measured.Continuous mode is identical as test case 1, and measurement result is detailed in table 2.Wherein, 40 patients combine iconography and pathology to measure, and clinical diagnosis obtains whether be breast cancer.By comparing Out, it is breast cancer feminine gender that CEA content, which is less than 100ng/mL, in the nipple discharge sample of patient, and CEA contains in nipple discharge sample It is breast cancer positive that amount, which is greater than or equal to 100ng/mL,.

The result that the test strips of 2 embodiment 3 of table are measured nipple discharge sample

From table 2 it can be seen that the test strips using embodiment 3 detect the nipple discharge of patient, examined with pathology The disconnected testing result obtained is consistent, illustrates that the CEA test strip of above embodiment can be used in detecting in nipple discharge CEA content, therefore, it is determined that whether suffering from breast cancer.

Test case 3

Using the CEA test strip of embodiment 3 and embodiment 8 respectively to the serum sample and cream of 60 breast cancer patients Head discharge is detected, and using CEA test strip, the CEA detection kit of Roche Holding Ag of embodiment 3 and embodiment 8 The serum sample of 60 Healthy Peoples is detected respectively, see Table 3 for details for testing result.Test method refers to test case 1, test mark Standard refers to test case 2.

The test result of table 3 breast cancer patients and Healthy People

From table 3 it can be seen that using the test strips of embodiment 3 to the serum sample and nipple discharge sample of breast cancer patients The recall rate of detection be higher than the test strips of embodiment 8, further illustrate that the detection of test strips that above embodiment obtains is quasi- True property and recall rate are higher.And do not detected in embodiment 8 there are 1~2, it may be possible to caused by low value sensitivity is lower.

Test case 4

Experiment is divided into two groups, respectively experimental group and control group.

In experimental group: being detected, obtained to the nipple discharge of different CEA concentration using the CEA test strip of embodiment 3 To the ratio (i.e. signal value) of detection line and the signal value of nature controlling line (C band), the content of CEA is lower than in above-mentioned nipple discharge 10ng/mL, continuous mode are identical as test case 1.See Table 4 for details for measurement result.

In control group, nipple discharge identical in experimental group is detected using the CEA test strip of embodiment 3, Obtain the ratio (i.e. signal value) of the signal value of detection line and nature controlling line.Continuous mode is roughly the same with test case 1, difference It is, well room temperature reaction is added after ten minutes in sample to be tested, ID chip is directly inserted into immunity analysis instrument, will test card It is inserted into immunity analysis instrument card inserting mouth, is clicked " test ", instrument calculates the dense of CEA in sample to be tested by analysis software automatically Degree.TMB is not added in control group to develop the color.See Table 4 for details for measurement result.

The measurement result of table 4 experimental group and control group

From table 4, it can be seen that at the same concentration, the signal value of experimental group is higher than the signal value of control group, and explanation passes through TMB color developing agent is added in above-mentioned test strips being capable of amplification detection signal.When sample concentration is 0.11ng/mL, experimental group Signal value is 0.18, illustrates the kit combination TMB color developing agent sensitivity with higher by above embodiment.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims

1. a kind of CEA test strip characterized by comprising bottom plate and the loading pad on the bottom plate, combination Pad, cushion, coated film and absorption pad, the loading pad, the bonding pad, the cushion, the coated film and institute It states absorption pad to be sequentially connected with from one end of the bottom plate to the other end, the bonding pad is equipped with the first CEA of nano enzyme label Monoclonal antibody, the coated film are equipped with detection line, and the detection line is equipped with the 2nd CEA monoclonal antibody, the nano enzyme The first CEA monoclonal antibody and the 2nd CEA monoclonal antibody of label can be in conjunction with the different epitopes of CEA.

2. CEA test strip according to claim 1, which is characterized in that the nano enzyme can be catalyzed 3,3', 5, The colour developing of 5'- tetramethyl benzidine.

3. CEA test strip according to claim 1, which is characterized in that the nano enzyme is magnetic ferroferric oxide Nano particle, the nano enzyme surface have conjugated group, and the conjugated group can be with the first CEA monoclonal antibody knot It closes.

4. CEA test strip according to claim 1, which is characterized in that be additionally provided with nano enzyme mark on the bonding pad The Avidin of note, the nature controlling line with the detection line interval is additionally provided on the coated film, and the nature controlling line is located at the detection Line is provided with Avidin antibody close to the side of the absorption pad, the nature controlling line.

5. a kind of preparation method of CEA test strip, which comprises the steps of:

2nd CEA monoclonal antibody is set on nitrocellulose filter, it is dry, the coated film with detection line is obtained, it is described 2nd CEA monoclonal antibody can combine different CEA epitopes from the first CEA monoclonal antibody that the nano enzyme marks；And

Loading pad, the bonding pad, cushion, the coating are sequentially connected with from one end of the bottom plate to the other end on bottom plate Film and absorption pad obtain CEA test strip.

6. the preparation method of CEA test strip according to claim 5, which is characterized in that described to mark nano enzyme The first CEA monoclonal antibody be set on polyester cellulose film before, further include the first CEA for preparing nano enzyme label The step of monoclonal antibody:

Nano enzyme, carbodiimide and n-hydroxysuccinimide are mixed and reacted, is separated by solid-liquid separation, precipitating is collected；It will be described heavy Shallow lake is reacted with the first CEA monoclonal antibody, obtains the first CEA monoclonal antibody that the nano enzyme marks, and described first The mass ratio of CEA monoclonal antibody and the nano enzyme is 0.1:1~1:1；

Alternatively, mixing and reacting nano enzyme with the first CEA monoclonal antibody, the first CEA for obtaining the nano enzyme label is mono- The mass ratio of clonal antibody, the first CEA monoclonal antibody and the nano enzyme is 0.2:1~2:1.

7. the preparation method of CEA test strip according to claim 6, which is characterized in that described into the precipitating It is added after the first CEA monoclonal antibody reacted, further includes following steps: to the precipitating and the first CEA Dan Ke Confining liquid is added in the reactant that grand antibody response obtains to be reacted, the confining liquid includes the PBS buffering of 0.01M~0.1M The bovine serum albumin bletilla mass percentage that liquid, mass percentage are 0.1%~1% is 0.01%~0.1% Triton X-100；

Alternatively, including the following steps: after the nano enzyme is mixed and reacted with the first CEA monoclonal antibody to the nanometer Confining liquid is added in the reactant that enzyme is obtained with the first CEA monoclonal antibody reactive to be reacted, the confining liquid includes 0.01M The bovine serum albumin bletilla mass percentage that the PBS buffer solution of~0.1M, mass percentage are 0.1%~1% is 0.01%~0.1% Triton X-100.

8. the preparation method of CEA test strip according to claim 6, which is characterized in that the first CEA monoclonal Antibody is secreted to obtain by hybridoma, the preparation step of the hybridoma are as follows: using spleen cell of the CEA after immune with Myeloma cell's fusion, obtains the hybridoma.

9. the preparation method of CEA test strip according to any one of claim 5~8, which is characterized in that described to incite somebody to action First CEA monoclonal antibody of nano enzyme label is set to the step on polyester cellulose film specifically: by the nano enzyme mark First CEA monoclonal antibody of note is mixed with treatment fluid, obtains antibody spray coating liquor；The antibody spray coating liquor is sprayed into described poly- On ester fiber element film；The treatment fluid includes the bovine serum albumin(BSA) that mass percentage is 0.1%~1%, quality percentage contains Amount for 5%~20% trehalose, mass percentage be 0.1%~2% casein, mass percentage be 0.1%~ The Tris buffer of pH7.4~9.0 of 1% S9 surfactant, 0.01M~0.2M；

And/or described 2nd CEA monoclonal antibody is set to the step on nitrocellulose filter specifically: use coating buffer After the concentration to 0.5mg/mL~2.0mg/mL for adjusting the 2nd CEA monoclonal antibody, then draw the nitrocellulose filter On, the coating buffer include the PBS buffer solution of 0.01M~0.05M, mass percentage be 5%~20% trehalose, quality The methanol that percentage composition is 1%~3%.

10. a kind of CEA detection device, which is characterized in that including the described in any item CEA test strips of Claims 1 to 4 or The CEA test strip that the preparation method of the described in any item CEA test strips of person's claim 5~9 is prepared.