CN108535495A - A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method quantitatively detecting CYFRA21-1 in blood - Google Patents

Abstract

The present invention relates to a kind of time-resolved fluoroimmunoassay chromatograph test strips and preparation method thereof quantitatively detecting CYFRA21 1 in blood.The test strips are the NC films in interlaced 2mm stickups successively on white PVC bottom plates, 1 antibody of CYFRA21 and the fluorescent microsphere conjugate pad of mouse anti-rabbit IgG is marked, sample pad, water absorption pad, then it is got stuck the test card that fixed test strips assemble with mating two plastics up and down again, 1 antibody detection lines of CYFRA21 and rabbit igg nature controlling line are surrounded by NC films in advance, time-resolved fluoroimmunoassay chromatographic technique has been introduced into blood in the quantitative detection of CYFRA21 1 by the present invention, detection time is not only greatly saved, improve stability and the sensitivity of detection, and it is easy to operate, it can be used for field screening；Simultaneously also with cheap, the cost-effective advantage of cost.

Description

A kind of time-resolved fluoroimmunoassay chromatography examination quantitatively detecting CYFRA21-1 in blood Paper slip and preparation method

Technical field

The invention belongs to field of medical examination, more particularly to a kind of time for quantitatively detecting CYFRA21-1 in blood point Distinguish fluorescence immune chromatography test paper bar and preparation method.

Background technology

CA15-3 is the most important Specific marker of breast cancer.The CA15-3 of the patient with breast cancer of 30%-50% is apparent It increases, the variation of content is closely related with therapeutic effect, is patient with breast cancer's diagnosis and monitoring postoperative recurrence, observation curative effect Optimal parameter.CA15-3 dynamics measure the early detection for helping to recur after II phases and III primary breast cancer Case treatments：When When CA15-3 is more than 100U/mL, it is believed that have metastatic lesion.The growth and decline of patients serum's CA15-3 levels and breast cancer disease end of love Change parallel, be the signal of interest of recurrence and transfer, and this signal is sent out than the appearance of clinical symptoms and with such as B The time of the detections such as super, x-ray or CT recurrence and transfer is early.It is reported that CA15-3 levels be more than 30U/mL, 40U/mL and When 50U/mL, judging patient with breast cancer, there are loco-recurrence after resection or DISTANT METASTASES INs, and sensibility is more than 90%, specifically Property is respectively 95%, 99% and 100%, and correct judgement rate respectively reaches 56%, 83% and 100%.In addition, the breast that CA15-3 levels increase The time that adenocarcinoma patients shift is early many compared with the normal patients of CA15-3.According to analysis and research, blood serum of patients with human breast carcinoma CA15-3 levels change the consistency for existing between its regional nodes and DISTANT METASTASES IN situation and changing, and especially have and turn at a distance Stove person is moved, CA15-3 expressions and positive rate dramatically increase, so, CA15-3 has plays monitoring to Metastasis in Breast Cancer Effect, if its lasting serum levels increases, then should start or reinforce chemotherapy, radiotherapy or use endocrine therapy instead etc..

The diagnostic techniques of CYFRA21-1 includes mainly in current blood：Enzyme linked immunological is tested（ELISA）, chemiluminescence （CLIA）, the methods of colloidal gold immunochromatographimethod, but these methods have the characteristics that respective and deficiency：ELISA and chemiluminescence with And time-resolved fluorescence method principle is similar, and it is only variant in terms of sensitivity, automation is mostly realized at present, high-volume, is determined The shortcomings of amount detects, but there are result difference between method is big, and the range of linearity is relatively narrow, in addition, automatic detection is at present by foreign countries Large manufacturer is monopolized, and instrument and equipment is expensive, and is not suitable for single part and small batch detection use, greatly limits them in base , there is colloidal gold immunity chromatography to detect total CYFRA21-1 in recent years in the application of layer hospital, clinic, quickly, convenient, single Person-portion operates, but a disadvantage is that sxemiquantitative can only be realized, can only indicate a probable ranges, cannot achieve accurate quantification, and Because of the limitation of colloidal gold sensitivity, low concentration CYFRA21-1 can not be detected, monstrosity is needed using other methods review inspection It surveys, virtually increases the medical treatment cost of testee, there is larger difficulty in terms of marketing, it is difficult to which large area uses.

Time resolved fluoro-immunoassay（TRFIA）It is that one kind for founding on the basis of conventional fluorescent is analyzed is novel non-to put Penetrating property immuno analytical method.TRFIA is to contain the nanoparticles of lanthanide series rare-earth elements as marker, according to lanthanide series metal chela It closes object fluorescence duration time length and Stokes displacements is big, measure fluorescence with TIME RESOLVED TECHNIQUE, effectively exclude non-specific fluorescence Interference, have the characteristics that high sensitivity, high specificity and stability are good.With classical time-resolved fluorescence immunoassay method DELFIA methods are different, time-resolved fluoroimmunoassay chromatographic technique using fluorescent nanometer microsphere as marker, can in each microballoon Thousands of a fluorescent moleculars are wrapped up, labeling effciency is substantially increased, effectively raises sensitivity；Nanometer fluorescent microspheres simultaneously Surface modification has the carboxyl of proper density, for the covalent coupling with albumen or antibody, improves the stability of marker.With section The development of skill, the immunochromatography technique in care diagnostic field, from first generation colloidal gold, color latex to second generation fluorescent microsphere Technology realizes the leap analyzed from qualitative to quantitative.And time-resolved fluoroimmunoassay chromatographic technique is then further, improves The stability of detection and sensitivity, and it is easy to operate, and detection time is short, can be used for field screening；Also there is cost just simultaneously Preferably, cost-effective advantage.

Time-resolved fluoroimmunoassay chromatography is the biology carried out with latex beads labelled antibody in immune response detection sample Active material provides the quantitative detection data to biological sample by detecting the fluorescence intensity that fluorescent nanometer microsphere is sent out, adopts With the standard curve of immune complex-fluorescence intensity of label, to calculate the amount of biological sample to be measured.Currently used mark Note latex beads are fluorescent nanometer microsphere, fluorescence will not be excited under the radiation situation of no specific wavelength ultraviolet light, only in spy Fluorescence can be just sent out under the ultra violet lamp of standing wave length, commercial fluorescence nanoparticle all have passed through the modification on surface, big generous Label process, label is easy, reproducible.

Invention content

The purpose of the present invention is to apply time-resolved fluoroimmunoassay chromatographic technique in CYFRA21-1 quantitative testing test papers In item, by CYFRA21-1 antibody and rabbit igg difference covalent coupling on fluorescent nanometer microsphere, the two is made after mixing in proportion For detection line and nature controlling line, the detection of sample is carried out according to the principle of routine immunization chromatography, it is glimmering in conjunction with easy-to-use dry type Light immunity analysis instrument is detected, and is being realized the window phase that can greatly shorten detection while highly sensitive detection again, can kept away Exempt from various drawbacks of aforementioned several detection methods, and combines the advantage of aforementioned several method：It can be detected with single room, it can also Batch detection, and accurate quantitative result can be provided immediately, measuring instrument is simple and reliable, easy to operate, convenient and practical.

For achieve the above purposes, technical scheme is as follows：It is a kind of quantitatively detect blood in CYFRA21-1 when Between resolved fluorometric immuno-chromatographic test paper strip, which includes close-connected sample pad 1, fluorescence successively on bottom plate 7 and bottom plate 7 Microballoon conjugate pad 2, reaction film 3 and water absorption pad 6.

Its bottom plate is PVC bottom plates；It is coated with CYFRA21-1 antibody on fluorescent microsphere conjugate pad and mouse anti-rabbit IgG is anti- Body；Reaction film is nitric acid vitamin（NC）Film, NC films are equipped with detection line 4 and nature controlling line 5；It is coated in detection line 4 CYFRA21-1 antibody is coated with rabbit igg on nature controlling line 5..

The wherein described conjugate pad is to pad the pretreated glass fibre for the treatment of fluid by conjugate, at the conjugate pad It is the M004 blocking agents containing 0.1mg/mL, 1% boric acid, the borate buffer solution of 0.5% BSA and 3% trehalose to manage liquid.

In embodiments of the invention, the sample pad 1, fluorescent microsphere conjugate pad 2, reaction film 3 and water absorption pad 6 exist Bottom plate 7 arranges in order, wherein the sample pad 1 partially overlaps with the fluorescent microsphere conjugate pad 2, intersection length Account for the fluorescent microsphere conjugate pads 2 length 1/5~1/3；The fluorescent microsphere conjugate pad 2 has part weight with reaction film 3 It closes, intersection length accounts for 1/5~1/3 that the fluorescent microsphere conjugate pads 2 length；The reaction film 3 has portion with water absorption pad 6 Divide and overlap, intersection length accounts for 1/10~1/5 that the fluorescent microsphere conjugate pads 2 length；The detection line 4 and nature controlling line 5 length accounts for the 1/15~1/10 of 3 length of the reaction film；The distance of the detection line 4 and sample pad 1 accounts for the reaction film 3 The 1/4~1/3 of length；Spacing between the detection line 4 and nature controlling line 5 accounts for the 1/3~1/2 of the reaction film.

In one particular embodiment of the present invention, the length of sample pad 1 is 20mm, the length of fluorescent microsphere conjugate pad 2 Degree is 6mm, and sample pad 1 overlaps 2mm with fluorescent microsphere conjugate pad；The length of reaction film 3 is 25mm, and reaction film 3 and fluorescence are micro- Ball conjugate pad overlaps 2mm；The length of water absorption pad 6 is 17mm, the coincidence 2mm of water absorption pad 6 and reaction film 3；Detection line 4 is apart from sample The width of this pad 7mm, detection line and nature controlling line is 2mm, and the two is at a distance of 6mm.

In one embodiment of the invention, final concentration of 0.05~0.5mg/mL of the CYFRA21-1 antibody.At this In one preferred embodiment of invention, final concentration of 0.1~0.3mg/mL of the CYFRA21-1 antibody.The one of the present invention In a most preferred embodiment, the final concentration of 0.2mg/mL of the CYFRA21-1 antibody.

In one embodiment of the invention, final concentration of 0.05~0.5mg/mL of the mouse anti-rabbit IgG antibody.At this In one preferred embodiment of invention, final concentration of 0.1~0.3mg/mL of the mouse anti-rabbit IgG antibody.The one of the present invention In a more preferred embodiment, 0.2 mg/mL of final concentration of the mouse anti-rabbit IgG antibody.

In one embodiment of the invention, a diameter of 50~500nm of the fluorescent microsphere.One in the present invention is excellent It selects in embodiment, a diameter of 100~300nm of the fluorescent microsphere.It is described glimmering in the more preferred embodiment of the present invention A diameter of 200nm of light microballoon.

In the implementation of the present invention is complete, the fluorescent microsphere is loaded with lanthanide series or its chelate, the group of the lanthanides member Element is preferably samarium, europium or terbium.

The present invention also provides the preparation methods of any of the above-described kind of time-resolved fluoroimmunoassay chromatograph test strip, including following step Suddenly：

A, the processing of conjugate pad：Conjugate pad treatment fluid is sprayed at conjugate with the amount of 16 μ L/cm with quantitative liquid-jet device It is dried 24 hours for 45 DEG C after on pad；

B, the preparation of NC films：Anti- CYFRA21-1 coated antibodies and rabbit igg are diluted to 0.8mg/ respectively using coating buffer solution ML and 1.0mg/mL concentration, using quantitative liquid-jet device respectively by the two with the interval spray printing of 0.5-0.8cm in nitrocellulose On film, after in 45 DEG C dry 72 hours, be added drier seal up for safekeeping it is spare；

C, the preparation of conjugate pad：The latex beads for selecting a diameter of 200nm, use carbodiimide（EDC）The side of covalent coupling CYFRA21-1 antibody and mouse anti-rabbit IgG are tagged on latex beads by formula；The latex beads prepared are filled using quantitative hydrojet It sets and is sprayed on processed conjugate pad with the amount of 8 μ L/cm；

D, the assembling of test strips：Sticked to interlaced 2mm successively on white PVC bottom plates NC films, conjugate pad, sample pad, Water absorption pad is to get to time-resolved fluorescence immuno-chromatographic test paper strip.

Further, above-mentioned step C includes following three step：

1）The preparation of fluorescent labeled antibody：The latex beads for selecting a diameter of 200nm are buffered using the MES containing 0.05mol/L Liquid washs latex beads, and EDC and NHS, which is added, to be made final concentration of to be respectively 0.5mg/mL and 5mg/mL, and ultrasonic mixing is set shaking table and kept away Light is incubated 15min, and centrifugation 20min removes supernatant, antibody is added after being washed twice with MES so that the final concentration of 0.2mg/mL of antibody, It is placed in shaking table and is protected from light incubation 3h, 30% confining liquid is added, be placed in shaking table and be protected from light incubation 1.5h, remove supernatant after the completion of closing, use MES It washes twice, 0.05% Tween-20, the preservation buffering of the 50mmol Tris-Hcl pH8.2 of 0.1% casein and 0.5%BSA Liquid redissolves latex beads, and 4 DEG C save backup；Mouse anti-rabbit IgG is tagged on latex beads using same method, rear knot Object dilution is closed with 1:10-1:15 ratio dilutes the CYFRA21-1 marked and mouse anti-rabbit IgG antibody.

Further, in above-mentioned step C, the spraying method of fluorescent microsphere antibody is：The fluorescent microsphere antibody that will have been diluted Mixture is sprayed at the amount of 8 μ L/cm on processed conjugate pad using quantitative liquid-jet device, and drier envelope is added after dry It deposits spare.

Further, in above-mentioned step B, the preparation method of NC films is：CYFRA21-1 antibody is diluted with coating buffer solution For 0.8mg/mL, goat anti-rabbit igg is diluted to 1mg/mL, using quantitative spray film device with the amount of 1 μ L/cm by the two with 0.6cm's Spray printing is spaced on nitrocellulose filter, after dried 72 hours in 45 DEG C, be added drier seal up for safekeeping it is spare.

Compared with existing Rapid detection test strip, the present invention has the following advantages：

1）By the improvement to test strips, time-resolved fluoroimmunoassay chromatographic technique is introduced into the detection of CYFRA21-1 for the first time, In conjunction with dry type fluorescence immunity analyzer, the quantitative detection of CYFRA21-1 is realized, is provided a great convenience for Clinical practice.

2）It realizes for the first time and detects CYFRA21-1 in a test strips, greatly facilitate clinical diagnosis.

The present invention is easy to operate, is suitble to large-scale production, detects required portable device and also listed, therefore can be wide It is general to use unit and some blood sampling scene, rural area and base clinics etc. for the high-volume such as hospital, blood station, epidemic prevention station, physical examination The unit that small lot or single part use uses.There is positive meaning for the quantitative Diagnosis of breast cancer.

Description of the drawings

Fig. 1 shows the test strips of CYFRA21-1 in time-resolved fluoroimmunoassay chromatography standard measure detection blood of the present invention Structural schematic diagram.

Fig. 2 shows a specific test strips structure schematic diagram of the invention.

Fig. 3 shows the double-log standard curve of invention detection card detection CYFRA21-1.

Specific implementation mode

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention be more clearly understood, below in conjunction with Embodiment, the present invention will be described in further detail.

Time-resolved fluoroimmunoassay chromatograph test strip such as Fig. 1 of the present invention for quantitatively detecting CYFRA21-1 in blood Shown in Fig. 2, which pastes to interlaced 2mm upper NC films 3 successively on bottom plate 7, combine CYFRA21-1 antibody and The test strips that conjugate pad 2, sample pad 1, the water absorption pad 6 of mouse anti-rabbit IgG assembles, it is pre-coated on NC films 3 to have detection line 4 （CYFRA21-1 antibody）With nature controlling line 5（Rabbit igg）.

In a particular embodiment, used CYFRA21-1 antibody is monoclonal antibody prepared by monoclonal antibody technique, and utilization is dual anti- The principle of the sandwich detection CYFRA21-1 antigens of body detects sample, when containing CYFRA21-1 antigens in sample to be measured, antigen meeting The antibody first and on conjugate pad being coupled combines, and with the progress that chromatography acts on, it is anti-that conjugate forward movement reaches CYFRA21-1 Body is coated at line 4, and antigen can be gathered in conjunction with double-antibody sandwich compound is formed at coating line 4 with coated antibody again, separately Outside, rabbit igg mark fluorescent microballoon antibody will continue to move ahead, reach nature controlling line 5 when, mouse anti-rabbit IgG can be combined with rabbit igg thus Equally occurs fluorescent microsphere aggregation at C lines.Entire reaction carries out complete in 30 minutes, and General reactions can be into after 15 minutes Machine-read card on row, detection line and nature controlling line can all generate corresponding fluorescence signal value, time-resolved fluoroimmunoassay chromatography detection Practical measured value can be substituted into preset standard curve by instrument according to the 2 D code information on detection card can obtain the result of the amount of determination. Measured value is substituted into the process complete sequencing that preset standard curve obtains quantitative values by entire Card Reader, identification Quick Response Code, is done Formula fluorescence immunity analyzer can directly give quantitative result.

Embodiment

Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under State the technology disclosed in example represent inventor discovery can be used for implement the present invention technology, therefore can be considered as implementation this The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.

Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.

Those skilled in the art will recognize or just will appreciate that by routine test many described here Invention particular embodiment many equivalent technologies.These will equally be comprised in claims.

Embodiment 1 quantitatively detects the time-resolved fluoroimmunoassay chromatograph test strip and kit of CYFRA21-1 in blood Preparation method

The preparation method of the present embodiment includes the following steps：

A, the preparation of NC films：

It is coated with the preparation of buffer solution：The PB buffer solutions and 1% trehalose of 0.05mol/L pH 7.4,0.22 μm of filtering with microporous membrane 4 DEG C of degerming postposition saves backup, the term of validity two weeks.

The coating of NC films：CYFRA21-1 antibody is diluted to 0.8mg/mL with coating buffer solution, rabbit igg is diluted to 1.0mg/mL, using quantitative spray film device with the amount of 1 μ L/cm by the two with the uniform spray printing in the interval of 0.6cm in 2.5cm width nitre On sour tunica fibrosa, after in 45 DEG C dry 72 hours, be added drier seal up for safekeeping it is spare.

B, the preparation of fluorescent microsphere antibody：

The preparation of the MES buffer solutions of 0.05mol/L：It is 6.0 with purified water and MES secure phs, the MES of a concentration of 50mmol/L Buffer solution saves backup for 4 DEG C after 0.22 μm of miillpore filter degerming, the term of validity two weeks.

The preparation of storing liquid：The Tris-HCl buffer solutions that final concentration 50mmol is prepared with purified water, dense HCl and Tris, add Enter BSA, Casein, Tween-20, final concentration is 0.5%, 0.1%, 0.05% respectively, 4 DEG C after 0.22 μm of filtering with microporous membrane degerming Save backup the term of validity two weeks.

The preparation of fluorescent labeled antibody：The latex beads for selecting a diameter of 200nm, it is slow using the MES containing 0.05mol/L Fliud flushing washs latex beads, and EDC and NHS, which is added, to be made final concentration of to be respectively 0.5mg/mL and 5mg/mL, and ultrasonic mixing sets shaking table It is protected from light and is incubated 15min, centrifugation 20min removes supernatant, antibody is added after being washed twice with MES so that the final concentration of 0.2mg/ of antibody ML is placed in shaking table and is protected from light incubation 3h, 30% confining liquid is added, and is placed in shaking table and is protected from light incubation 1.5h, removes supernatant after the completion of closing, use MES is washed twice, 0.05% Tween-20, the preservation of the 50mmol Tris-Hcl pH8.2 of 0.1% casein and 0.5%BSA Buffer solution redissolves latex beads, and 4 DEG C save backup；Mouse anti-rabbit IgG is tagged on latex beads using same method, after With conjugate dilution with 1:10-1:15 ratio dilutes the CYFRA21-1 marked and mouse anti-rabbit IgG antibody.

C, the processing of conjugate pad

With 45 DEG C after conjugate pad treatment fluid is sprayed at the amount of 16 μ L/cm on the glass fibre of 6mm with quantitative liquid-jet device Drying 24 hours.

Conjugate pad treatment fluid is the M004 blocking agents containing 0.1mg/mL, 1% boric acid, the seas 0.5% BSA and 3% The borate buffer solution of algae sugar.

D, the spraying and drying of fluorescent microsphere antibody

Using IsoFlow spray film instrument it is nozzle specially used by prepared fluorescent microsphere mixture on the amount even application of 8 μ L/cm On the processed fiberglass packing of 0.6cm width, 45 DEG C of dry 72h, addition drier is sealed up for safekeeping spare；

E, the assembling and cutting of test strips

Following all operations all must be less than 30% in humidity, be carried out in the room of 20-25 DEG C of temperature.

The assembling of test paper plate：As requested by 2.5cmNC films, 1.7cm blotting papers, 0.6cm conjugate pads, 2.0cm samples Pad is assembled on 6.0cm width white PVC bottom plates, is assembled into test paper plate.

Test strips are cut：Assembled test paper plate is cut into 0.4cm's wide using 4000 type cutting machines of BioDot CM Finished product test strips.

F, the assembling of test card

Single part test strips of well cutting of the present invention are placed in the card slot on plastics Negative card, upper cover is covered, are used Card press machine compresses upper and lower two panels plastic clip, it is ensured that entire test strips are in tensioned state.Addition drier room temperature is sealed up for safekeeping spare.

G, the 2 D code information of the batch is determined

The name of an article：Non-small cell carcinoma associated antigen 19-9 segment（CYFRA21-1）Detection kit（Time-resolved fluoroimmunoassay chromatographs Method）

Batch：The assembling date of test card, format are：Year/Month/Day, XXX/XX/X

H, the printing of Quick Response Code

Pattern in 2 D code comprising project name and lot number is sprayed to the specific position of test card upper cover by laser marker, Using dry type fluorescence immunity analyzer, sampling observation 2% ensures that Quick Response Code reading is errorless at random.

I, finished product packing

Single part test card of spray coated Quick Response Code and a drying prescription of being responsible for a task until it is completed are sealed in aluminium foil bag, 20 person-portions are a bag apparatus In one packing box, a box portion specification and an ID chip with standard curve are kept in dark place in room temperature, and the shelf-life is 18 months.

Embodiment 2

In addition to fluorescent microsphere antibody preparation the step of in：The molecular proportion that CYFRA21-1 antibody and latex beads is added is 10:1. Other steps are the same as embodiment 1.

Embodiment 3

In addition in the mixing step of fluorescent microsphere antibody：By the two using conjugate dilution with 1:10 ratio is dilute by mixture It releases fiberglass packing to be sprayed to use, other steps are the same as embodiment 1.

Embodiment 4

In addition in the mixing step of the fluorescent microsphere marked：By the two using conjugate dilution with 1:15 ratio will mix Object dilutes fiberglass packing to be sprayed and uses, and other steps are the same as embodiment 1.

5 present invention detection card standard curve of embodiment is established

CYFRA21-1 standard antigen raw materials are taken, are demarcated with chemoluminescence method, 300ng/ is diluted to normal human serum liquid ML, 150ng/mL, 50ng/mL, 10ng/ mL, 3.3ng/ mL, 1ng/mL, each standard point makees 10 tests, according to statistics Method, which determines the relationship of each standard point and fluorescence measurement value and establishes equation, is allowed to be fitted linear, this equation and standard curve Dry type fluorescence immunity analyzer can be made to be determined sample measured value.

Standard curve is drawn according to double logarithm method, as shown in Figure 3.The result shows that fluorescence-CYFRA21-1 concentration double-logs Curve correlation coefficient R reaches 0.9993, has extraordinary linear relationship.

The application method of the detection card of 6 present invention of embodiment

1, it is loaded

The detection card of single part is taken out from packing box, test paper is placed on smooth desktop, use is micro by this card packaging of aluminium foil bag Pipettor takes 70 μ L serum samples to be added in the well on card, and reaction is waited for carry out 15 minutes.

2, it measures and result exports

ID chips are inserted into ID bayonets first, ID chips are then read on instrument, then detection card insertion is entered into dry type fluorescence immunoassay The card inserting mouth of analyzer runs instrument, and instrument can voluntarily read the 2 D code information of card, measures and printing measures knot immediately Fruit, quantitative result can be shown in print result.

The sensitivity of 7 present invention detection card of embodiment

Prepare the test solution of various concentration using CYFRA21-1 standard items, specific a concentration of 0.25,0.5,1,2,5,10,20 μg/L。

Concentration test solution is added drop-wise to the sample of time-resolved fluoroimmunoassay chromatography detection card prepared by embodiment 1-4 respectively On product pad, 5-8min is reacted, test strips are then put into simple fluorescence detector detection window, in 300~400nm light source activations Under, observe result.

The result shows that detection card sensitivity prepared by embodiment 1-4 is splendid, reach<0.25μg/L.

The stability of 8 present invention detection card of embodiment

The embodiment 1-4 detections prepared are stuck in and place 3,6 and 18 months at room temperature, are then carried out according to the method for embodiment 7 Sensitivity test.

The result shows that（Table 1）, after detection card is stored 3 months, the sensitivity of detection card prepared by embodiment 1-4 is still whole Reach<0.25μg/L.Detection card is deposited after six months, and detection card sensitivity prepared by embodiment 2 and embodiment 3 is declined, and is <0.5 μ g/L, but detection card sensitivity prepared by embodiment 1 and embodiment 4 still reaches<0.25μg/L.Detection card storage 18 After month, detection card sensitivity prepared by embodiment 2 drops to<1 μ g/L, detection card sensitivity prepared by embodiment 4 drop to<2μ G/L, and detection card sensitivity prepared by embodiment 1 and embodiment 3 still can reach<0.5μg/L.

The detection card of table 1 places 3,6 and sensitivity results after 12 months

	Embodiment 1	Embodiment 2	Embodiment 3	Embodiment 4
					3 months	<0.25μg/L	<0.25μg/L	<0.25μg/L	<0.25μg/L
6 months	<0.25μg/L	<0.5μg/L	<0.5μg/L	<0.25μg/L
					12 months	<0.5μg/L	<1μg/L	<0.5μg/L	<2μg/L

The precision of 9 present invention detection card of embodiment

The detection card prepared using the present embodiment 1-4 is detected as CYFRA21-1 feminine genders to 100 parts through efficient liquid phase tandem mass spectrum Sample is detected according to the present invention using qualitative checking method under room temperature, and observation testing result is judged.

Experimental result is the positive beyond detection limit, is feminine gender without departing from detection limit.

The result shows that in 100 parts of negative samples, the detection card prepared using embodiment 1 detects 100 parts of negative findings, 0 part of false positive, false positive rate 0%, accuracy rate 100%.Negative findings 99 are detected using the embodiment 2-3 detection cards prepared Part, 1 part of false positive, false positive rate 1%, accuracy rate 99%.The detection card prepared using embodiment 4 detects negative findings 98 Part, 2 parts of false positive, false positive rate 2%, accuracy rate 98%.

Equally, a concentration of 0.5 μ g/L samples of 100 parts of CYFRA21-1 of detection card pair prepared using the present embodiment 1-4, room It is detected using qualitative checking method according to the present invention under the conditions of temperature, observation testing result is judged.

The result shows that in 100 parts of negative samples, the detection card prepared using embodiment 1 detects 100 parts of positive findings, 0 part of false negative, false negative rate 0%, accuracy rate 100%.The detection card prepared using embodiment 2 detects positive findings 98 Part, 2 parts of false negative, false negative rate 2%, accuracy rate 98%.The detection card prepared using embodiment 3 and 4 detects positive knot 99 parts of fruit, 1 part of false negative, false negative rate 1%, accuracy rate 99%.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (10)

1. a kind of time-resolved fluoroimmunoassay chromatograph test strip quantitatively detecting CYFRA21-1 in blood, which is characterized in that including Bottom plate, cover NC films successively on the bottom plate, CYFRA21-1 antibody is marked and the fluorescent microsphere conjugate pad of mouse anti-rabbit IgG, Sample pad and water absorption pad, the NC films are equipped with detection line and nature controlling line；CYFRA21-1 antibody is coated in the detection line, It is coated with rabbit igg on the nature controlling line.

2. time-resolved fluoroimmunoassay chromatograph test strip according to claim 1, which is characterized in that the test strips utilize Mating two plastic clips up and down are fixed.

3. time-resolved fluoroimmunoassay chromatograph test strip according to claim 2, which is characterized in that it is described get stuck on contain Quick Response Code, including project name and lot number information.

4. time-resolved fluoroimmunoassay chromatograph test strip according to claim 1, which is characterized in that the sample pad, glimmering Light microballoon conjugate pad, NC films and water absorption pad arrange in order in bottom plate, wherein the both ends of the fluorescent microsphere conjugate pad point Not with the interlaced 2mm in one end of one end of the sample pad and NC films, one end of the water absorption pad is another with the NC films Hold interlaced 2mm.

5. time-resolved fluoroimmunoassay chromatograph test strip according to claim 1, which is characterized in that the CYFRA21-1 The final concentration of 0.2mg/mL of antibody, the final concentration of 0.2mg/mL of the mouse anti-rabbit IgG antibody.

6. time-resolved fluoroimmunoassay chromatograph test strip according to claim 1, which is characterized in that the fluorescent microsphere A diameter of 200nm.

7. according to any time-resolved fluoroimmunoassay chromatograph test strips of claim 1-6, which is characterized in that the fluorescence Microballoon is loaded with lanthanide series or its chelate, and the lanthanide series is preferably samarium, europium or terbium.

8. according to the preparation method of any time-resolved fluoroimmunoassay chromatograph test strips of claim 1-6, feature exists In including the following steps：

A, the processing of conjugate pad：Conjugate pad treatment fluid is sprayed at conjugate with the amount of 16 μ L/cm with quantitative liquid-jet device It is dried 24 hours for 45 DEG C after on pad；

B, the preparation of NC films：Anti- CYFRA21-1 coated antibodies and rabbit igg are diluted to 0.8mg/ respectively using coating buffer solution ML and 1.0mg/mL concentration, using quantitative liquid-jet device respectively by the two with the interval spray printing of 0.5-0.8cm in nitrocellulose On film, after in 45 DEG C dry 72 hours, be added drier seal up for safekeeping it is spare；

C, the preparation of conjugate pad：The latex beads for selecting a diameter of 200nm, use carbodiimide（EDC）The side of covalent coupling CYFRA21-1 antibody and mouse anti-rabbit IgG are tagged on latex beads by formula；The latex beads prepared are filled using quantitative hydrojet It sets and is sprayed on processed conjugate pad with the amount of 8 μ L/cm；

D, the assembling of test strips：Sticked to interlaced 2mm successively on white PVC bottom plates NC films, conjugate pad, sample pad, Water absorption pad is to get to time-resolved fluorescence immuno-chromatographic test paper strip.

9. the preparation method of time-resolved fluoroimmunoassay chromatograph test strip according to claim 8, which is characterized in that described CYFRA21-1 antibody and mouse anti-rabbit IgG, which are tagged to the step on latex beads, in step C is：Using containing 0.05mol/L's MES buffer solutions wash latex beads, and EDC and NHS, which is added, to be made final concentration of to be respectively 0.5mg/mL and 5mg/mL, and ultrasonic mixing is set Shaking table, which is protected from light, is incubated 15min, and centrifugation 20min removes supernatant, antibody is added after being washed twice with MES so that antibody is final concentration of 0.2mg/mL is placed in shaking table and is protected from light incubation 3h, 30% confining liquid is added, and is placed in shaking table and is protected from light incubation 1.5h, is gone after the completion of closing Supernatant is washed twice with MES, 0.05% Tween-20, the 50mmolTris-Hcl pH8.2 of 0.1% casein and 0.5%BSA Preservation buffer solution, redissolve latex beads, 4 DEG C save backup；Mouse anti-rabbit IgG is tagged to latex beads using same method On, afterwards with conjugate dilution with 1:10-1:15 ratio dilutes the CYFRA21-1 marked and mouse anti-rabbit IgG antibody.

10. the preparation method of time-resolved fluoroimmunoassay chromatograph test strip according to claim 9, which is characterized in that also Including cutting reagent strip with according to the width that gets stuck, mating two plastics up and down fixed test strips that get stuck is used in combination to obtain test card Step.