CN109342719A - A kind of preparation method of thyroid function multi-link detection reagent kit - Google Patents
A kind of preparation method of thyroid function multi-link detection reagent kit Download PDFInfo
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- CN109342719A CN109342719A CN201811506408.0A CN201811506408A CN109342719A CN 109342719 A CN109342719 A CN 109342719A CN 201811506408 A CN201811506408 A CN 201811506408A CN 109342719 A CN109342719 A CN 109342719A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to field of biotechnology, more particularly to a kind of thyroid function multi-link detection reagent kit and preparation method thereof, the kit includes redissolving reagent, concentration washing lotion, enhancement solution, solid phase reaction cup, calibration curve card, calibration object, quality-control product and tracer；Redissolving reagent includes: the bovine serum albumin(BSA) of 10g/L~15g/L；Concentration washing lotion includes: the Tween-20 of 4.0ml/L~7.0ml/L；Enhancement solution includes: the three n-octyl phosphorous oxides of 3~4mg/L β-naphthoyltrifluoroacetone, 20~25mg/L；Solid phase reaction cup includes: the coating buffer of 1~10 μ g/ml, confining liquid；Tracer includes: target antibody or antigen conjugates, lanthanide ion chelate, tracer dilution.Kit of the present invention can realize five item detections, be that one kind is easy to operate, can be suitably used for the thyroid function detection kit of entry joint-detection, and have the characteristics that stability is good, detection limit is low, specific high, accuracy and precision are high.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of thyroid function multi-link detection reagent kit and its preparation
Method.
Background technique
Time resolution immunofluorescence assay technology is the novel ultramicron immunoassay skill that early eighties come out
Art.The TIME RESOLVED TECHNIQUE of fluorescence is the fluorescence lifetime difference using fluorescent emitter, by the separated fluorescence spectrum skill of its fluorescence
Art.Radioactive isotope is replaced using lanthanide series by tracer, with the lanthanide series with bifunctional group come labelled antigen or
Antibody, when the lanthanide series that disintegrates down and special amplification liquid form new chelate, fluorescence is remarkably reinforced and lasting.
The background fluorescence of sample itself can be eliminated with the method for delay measurements, to greatly improve detection sensitivity.
It is looked into newly according to State Food and Drug Administration website, has obtained the country registration same methodology of official written reply
Thyroid function detection kit, tracer is independent packaging, experiment when it is ready-to-use.
Currently, the thyroid function detection kit developed with time resolution detection technique, is mostly single detection
Mesh, and the often 3-8 index joints of making a definite diagnosis of disease are judged.In automation equipment in use, the detection of a sample, often
It needs to tear open plurality of reagents box and carries out jigsaw operation, and independently prepare corresponding tracer working solution not only due to introducing with container
And pollute, it is also easy to obscure when preparing and causes the failure of an experiment；In addition, needing to have more than needed in liquid feeding process, will lead to
Practical preparation tracer working solution is more than theoretical amount, and the surplus capacity of automation equipment is then more, therefore producer provides tracer
Object dosage is often more more than actual amount, and automation equipment is then higher, and the liquid more than needed tested every time discards, and will cause
Reagent waste.
Therefore, a kind of thyroid function detection kit easy to operate, can be suitably used for entry joint-detection will be effective
It solves the above problems.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of thyroid function multi-link detection reagent kit and its preparation sides
Method, in terms of properties of product, the kit has that stability is good, detection limit is low, linear relationship is good, high specificity, accurate
The features such as degree and precision are high.
A kind of present invention thyroid function multi-link detection reagent kit to be provided, gives reality by following technical solution
It is existing:
A kind of thyroid function multi-link detection reagent kit, the kit include redissolving reagent, concentration washing lotion, enhancing
Liquid, solid phase reaction cup, calibration curve card, calibration object, quality-control product and tracer.
As a preferred embodiment:
The redissolution reagent includes: bovine serum albumin(BSA), the buffer 1 of 10g/L~15g/L；
The concentration washing lotion includes: Tween-20, the buffer 2 of 4.0ml/L~7.0ml/L；
The enhancement solution includes: 3~4mg/L β-naphthoyltrifluoroacetone, 20~25mg/L three n-octyl phosphorous oxides,
Buffer 3；
The solid phase reaction cup includes: the coating buffer of 1~10 μ g/ml, confining liquid；Wherein the coating buffer by 1~
10ppm is coated with buffer and sterling target antibody or antigen conjugates dilution is made；
The calibration object is the calibration object for spending hormone negative serum and antigen sterling being prepared into 5 concentration；
The quality-control product is the quality-control product for spending hormone negative serum and antigen sterling being prepared into 3 concentration；
The tracer includes: target antibody, lanthanide ion chelate, tracer dilution.
As a preferred embodiment, the buffer 1 includes: the disodium ethylene diamine tetraacetate of 0.012~0.015g/L,
53~60mmol/L, pH7.8~8.0 Tris-HCl solution.
As a preferred embodiment, the buffer 2 includes: 0.405~0.425mol/L NaCl solution, 0.35~
The Tris-HCl buffer of 0.45mol/L, pH7.8~8.0.
As a preferred embodiment, the buffer 3 includes: 1.5~2ml/L TritonX-100,0.15~
0.2mol/L Potassium Hydrogen Phthalate-glacial acetic acid solution.
As a preferred embodiment, the confining liquid includes: 6~9.0g/L trehalose, 0.5~0.8%BSA, and 0.05
The sodium carbonate-bicarbonate buffer of~0.1mol/L, pH9.5~9.7.
As a preferred embodiment, the coating buffer includes: 6~8ppm Proclin300,0.07~
The sodium carbonate-bicarbonate buffer of 0.1mol/L, pH9.6.
As a preferred embodiment, the lanthanide ion chelate is Eu3+-N2[P- isocyanic acid-benzyl]-two
Ethylene triamine tetraacethyl sodium.
As a preferred embodiment, the tracer dilution includes following parts by weight of component: polysorbate40 0.3~0.5
Part, 0~3 part of Macrogol 6000,0~10 part of calf serum, ANS0~0.25 part, 0~0.3 part of sodium salicylate, BSA
0~5 part of Tris-HCl buffer.
As a preferred embodiment, the preparation method of the thyroid function multi-link detection reagent kit, specifically comprising as follows
Step:
(1) it redissolves the preparation of reagent: the bovine serum albumin(BSA) of 10g/L~15g/L being added in buffer 1；
(2) preparation of washing lotion is concentrated: the Tween-20 of 4.0ml/L~7.0ml/L being added in buffer 2；
(3) β-naphthoyltrifluoroacetone, the 20~25mg/L of 3~4mg/L the preparation of enhancement solution: are added into buffer 3
Three n-octyl phosphorous oxides；
(4) preparation of solid phase reaction cup: by the coating buffer of 1~10ppm of sterling target antibody or antigen conjugates
It is diluted to the coating buffer that concentration is 1~10 μ g/ml；Addition 100~200 hole μ l/ of coating buffer in porous microplate blank plate, 25~
It is incubated overnight under the conditions of 37 DEG C；The reaction cup being incubated for is patted dry with work cleaning solution board-washing 1~2 time；By washed reaction cup
Confining liquid is added by 200~280 holes μ l/, is incubated overnight and dries under the conditions of 23~27 DEG C；It is sealed with valve bag；Set 2~8 DEG C of items
It is saved under part；
(5) preparation of calibration object: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 5 concentration；
(6) preparation of quality-control product: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 3 concentration；
(7) Eu of 1mg the preparation of tracer: is added in 1mg~3mg target antibody3+-N2[P- isocyanic acid-benzyl]-two
It is mixed in ethylene triamine tetraacethyl sodium, 4~35 DEG C of 20~48h of reaction；Reaction solution is used through Sephadex G-50 column (1 × 40cm)
The Tris-HCl buffer of 50mmol/L, pH7.8 elute, separation tracer conjugate and free Eu3+, using full-automatic portion collection
Device carries out liquid collection with the speed of 1ml/ pipe；In carrying out fluorimeter number on fluorescence calculating instrument, collects fluorescence in first peak and count
Eluent greater than 10000000,2~8 DEG C of preservations after mixing；Europium mark working liquid concentration is determined using Checkerboard titration method, then
With tracer diluted, then it is lyophilized into microballoon, the microballoon after freeze-drying is transferred in (4) in solid phase reaction cup, sealed, then
It is transferred in aluminium plastic bag, is saved in 2~8 DEG C；
(8) by above-mentioned redissolution reagent, concentration washing lotion, enhancement solution, solid phase reaction cup, calibration curve card, calibration object, Quality Control
Product and tracer packing, labelling, the assembling for completing thyroid function multi-link detection reagent kit.
As a preferred embodiment, above-mentioned target antibody conjugate is that thyroxine monoclonal antibody or thyroid swash
Plain monoclonal antibody or trilute monoclonal antibody；Target antigen conjugate be thyroxine antigen conjugates or
Thyrotropic hormone conjugate or trilute antigen conjugates.
The utility model has the advantages that thyroid function multi-link detection reagent kit of the invention include molten reagent, concentration washing lotion, enhancement solution,
Solid phase reaction cup, calibration curve card, calibration object, quality-control product and tracer, can be to thyroxine, rush first shape by each part mentioned above
Glandular hormone, trilute, free triiodothyronine and free thyroxine carry out quantitative detection；It is obtained
Kit have the characteristics that stability it is good, detection limit it is low, linear relationship is good, high specificity, accuracy and precision it is high；The present invention
Every kind of reagent, material in the thyroid function multi-link detection reagent kit are required by accurate strict scientific matching,
The effect integrated can be only achieved excellent effect of the present invention；In addition, compared with existing market product, the present invention one
A kit can realize five item detections, be a kind of thyroid function that is easy to operate, can be suitably used for entry joint-detection
Detection kit.
Detailed description of the invention
Fig. 1,2,3,4,5 are invention kit and internal reagent box pattern detection linear dependence figure.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.
A kind of thyroid function multi-link detection reagent kit of embodiment
The preparation method of the thyroid function multi-link detection reagent kit, specifically includes the following steps:
(1) it redissolves the preparation of reagent: the bovine serum albumin(BSA) of 12g/L being added in buffer 1；The buffer 1 includes:
The disodium ethylene diamine tetraacetate of 0.014g/L, the Tris-HCl solution of 58mmol/L, pH7.9；
(2) preparation of washing lotion is concentrated: the Tween-20 of 5ml/L being added in buffer 2；The buffer 2 includes:
0.415mol/L NaCl solution, the Tris-HCl buffer of 0.4mol/L, pH7.8；
(3) preparation of enhancement solution: be added into buffer 3 β-naphthoyltrifluoroacetone of 4.5mg/L, 23mg/L three just
Octyl phosphorous oxide；The buffer 3 includes: 1.8ml/L TritonX-100,0.17mol/L Potassium Hydrogen Phthalate-ice vinegar
Acid solution；
(4) preparation of solid phase reaction cup: it is 5 μ g/ that sterling target antibody, which is diluted to concentration with the coating buffer of 6ppm,
The coating buffer of ml；It is incubated overnight under the conditions of 30 DEG C of 150 hole μ l/ of coating buffer is added in porous microplate blank plate；What is be incubated for consolidates
Phase reaction cup is patted dry with work cleaning solution board-washing 2 times；By washed solid phase reaction cup by 270 holes μ l/ be added confining liquid, 25
It is incubated overnight under the conditions of DEG C；Drying, dries overnight, is sealed with valve bag, saved under the conditions of setting 4 DEG C；
The coating buffer includes: the sodium carbonate-bicarbonate of 7ppm Proclin300,0.08mol/L, pH9.6
Buffer；
(5) preparation of calibration object: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 5 concentration；
(6) preparation of quality-control product: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 3 concentration；
(7) Eu of 1mg the preparation of tracer: is added in 2mg target antigen3+-N2[P- isocyanic acid-benzyl]-divinyl three
It is mixed in amine tetraacethyl sodium, 25 DEG C of reactions are for 24 hours；Reaction solution through Sephadex G-50 column (1 × 40cm) 50mmol/L,
The Tris-HCl buffer of pH7.8 elutes, separation tracer conjugate and free Eu3+, use full-automatic fraction collector with 1ml/
The speed of pipe carries out liquid collection；In carrying out fluorimeter number on fluorescence calculating instrument, collects fluorescence counting in first peak and be greater than
10000000 eluent, 4 DEG C of preservations after mixing；Europium mark working liquid concentration is determined using Checkerboard titration method, then uses tracer
Object diluted, then it is lyophilized into microballoon, the microballoon after freeze-drying is transferred in (4) in solid phase reaction cup, seals, transfers to
In aluminium plastic bag, saved in 4 DEG C；
(8) by above-mentioned redissolution reagent, concentration washing lotion, enhancement solution, solid phase reaction cup, calibration curve card, calibration object, Quality Control
Product and tracer packing, labelling, the assembling for completing thyroid function multi-link detection reagent kit.
The kit provided according to the present invention, using time-resolved fluoroimmunoassay technology, concrete operation method includes following
Step:
S1. the required amount of micropore solid phase reaction cup item of reagent is balanced to room temperature, then above-mentioned concentration washing lotion and go from
1:20 is mixed into work cleaning solution to sub- water by volume, and acquired work cleaning solution can also be used for coating preparation process；If desired
Using calibration object or Quality Control hole, need to redissolve calibration object, quality-control product with purified water before use；
S2. it is separately added into 150 μ l into every hole and redissolves reagent, then sequentially adds 50 μ l calibration objects or quality-control product or to be measured
Sample；Reaction system is at 37 DEG C, and oscillation incubation 15 minutes；With above-mentioned work cleaning solution board-washing 6 times；It is added into every hole
150 μ l enhancement solutions, slow oscillation 5min, in progress fluorescence counting in Fluorescent reader；It is fitted analysis by location parameter, is obtained
To quantitative result.
Kit of the present invention is identified according to methodology, can reach following index:
(1) minimum detection limit: the minimum detection limit of thyrotropic hormone of the present invention is not higher than 0.25 μ U/ml；Thyroxine
Minimum detection limit be not higher than 10mmol/L；The minimum detection limit of free thyroxine is not higher than 10mmol/L；Triiodo thyroid gland
The minimum detection limit of former propylhomoserin is not higher than 10mmol/L；The minimum detection limit of free triiodothyronine is not higher than
10mmol/L。
(2) linear: in linearly interval, the related coefficient (r) of five indices is not less than 0.9900.
(3) accuracy: detecting enterprise's calibration object that national standard is prepared within the scope of the linearly interval as defined in kit,
The relative deviation of its measurement result is in ± 10% range.
(4) withinrun precision: the high and low quality-control product being measured in parallel within the scope of kit, the coefficient of variation of measurement result
(CV) it is not higher than 10%.
(5) betweenrun precision: the high and low Quality Control between 3 different batches of product, within the scope of parallel determination kit
The coefficient of variation (CV) of product, measurement result is not higher than 15%.
(6) specific: thyrotropin assay 250U/L lutropin, 250U/L follicle-stimulating hormone (FSH), 10000U/L
The sample apparent concentration of human chorionic gonadtropin is not higher than 0.25 μ U/ml；Trilute and thyroxine, anti-three
The cross reacting rate of iodine thyronine is not higher than 5%；Thyroxine and trilute, anti-triiodo thyroid gland
The cross reacting rate of former propylhomoserin is not higher than 5%；The cross reacting rate of free thyroxine and free triiodothyronine
Not higher than 5%.
(7) stability: kit is placed 10 days under the conditions of 37 DEG C, and above-mentioned performance indicator still conforms to require.
(8) Fig. 1~5 are detection kit of the present invention and domestic contrast agents box pattern detection linear dependence figure, from Fig. 1
~5 as can be seen that the pattern detection result of kit of the present invention and the pattern detection result of domestic contrast agents box have height
Consistency.
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 (10)
1. a kind of thyroid function multi-link detection reagent kit, which is characterized in that the kit includes redissolving reagent, being concentrated and wash
Liquid, enhancement solution, solid phase reaction cup, calibration curve card, calibration object, quality-control product and tracer.
2. thyroid function multi-link detection reagent kit according to claim 1, it is characterised in that:
The redissolution reagent includes: bovine serum albumin(BSA), the buffer 1 of 10g/L~15g/L；
The concentration washing lotion includes: Tween-20, the buffer 2 of 4.0ml/L~7.0ml/L；
The enhancement solution includes: 3~4mg/L β-naphthoyltrifluoroacetone, the three n-octyl phosphorous oxides of 20~25mg/L, buffering
Liquid 3；
The solid phase reaction cup includes: the coating buffer of 1~10 μ g/ml, confining liquid；Wherein the coating buffer is by 1~10ppm
It is coated with buffer sterling target antibody or antigen conjugates dilution are made；
The calibration object is the calibration object for spending hormone negative serum and antigen sterling being prepared into 5 concentration；
The quality-control product is the quality-control product for spending hormone negative serum and antigen sterling being prepared into 3 concentration；
The tracer includes: target antibody, lanthanide ion chelate, tracer dilution；
The target antibody conjugate is thyroxine monoclonal antibody or thyrotropic hormone monoclonal antibody or triiodo first
Shape gland original ammonia acid monoclonal antibody；Target antigen conjugate be thyroxine antigen conjugates or thyrotropic hormone conjugate or
Trilute antigen conjugates.
3. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the buffer 1 includes:
The disodium ethylene diamine tetraacetate of 0.012 ~ 0.015g/L, 53 ~ 60mmol/L, pH7.8 ~ 8.0 Tris-HCl solution.
4. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the buffer 2 includes:
0.405 ~ 0.425mol/L NaCl solution, 0.35 ~ 0.45mol/L, pH7.8 ~ 8.0 Tris-HCl buffer.
5. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the buffer 3 includes:
1.5 ~ 2ml/L TritonX-100,0.15 ~ 0.2mol/L Potassium Hydrogen Phthalate-glacial acetic acid solution.
6. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the confining liquid includes: 6
~ 9.0g/L trehalose, 0.5 ~ 0.8%BSA, 0.05 ~ 0.1mol/L, pH9.5 ~ 9.7 sodium carbonate-bicarbonate buffer.
7. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the coating buffer packet
Contain: the sodium carbonate-bicarbonate buffer of 6 ~ 8 ppm Proclin300,0.07 ~ 0.1mol/L, pH9.6.
8. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the lanthanide ion
Chelate is Eu3+-N2[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium.
9. thyroid function multi-link detection reagent kit according to claim 2, which is characterized in that the tracer dilution
Include following parts by weight of component: 0.3 ~ 0.5 part of polysorbate40,0 ~ 3 part of Macrogol 6000,0 ~ 10 part of calf serum, ANS0 ~ 0.25
Part, 0 ~ 5 part of Tris-HCl buffer of 0 ~ 0.3 part of sodium salicylate, BSA.
10. thyroid function multi-link detection reagent kit according to claim 1, which is characterized in that the thyroid function is more
The preparation method of link detection reagent kit, specifically includes the following steps:
(1) it redissolves the preparation of reagent: the bovine serum albumin(BSA) of 10g/L~15g/L being added in buffer 1；
(2) preparation of washing lotion is concentrated: the Tween-20 of 4.0ml/L~7.0ml/L being added in buffer 2；
(3) preparation of enhancement solution: be added into buffer 3 β-naphthoyltrifluoroacetone of 3~4mg/L, 20~25mg/L three
N-octyl phosphorous oxide；
(4) preparation of solid phase reaction cup: sterling target antibody or the antigen conjugates coating buffer of 1~10ppm are diluted
The coating buffer for being 1~10 μ g/ml at concentration；100~200 hole μ l/ of coating buffer, 25 ~ 37 DEG C of conditions are added in blank reaction cup
Lower overnight incubation；Washed solid phase reaction cup is pressed 200 by the solid phase reaction cup work cleaning solution being incubated for board-washing 1 ~ 2 time
Confining liquid is added in~280 holes μ l/, is incubated overnight and dries under the conditions of 23 ~ 27 DEG C；It is sealed with valve bag；It is protected under the conditions of setting 2~8 DEG C
It deposits；
(5) preparation of calibration object: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 5 concentration；
(6) preparation of quality-control product: spending hormone negative serum and antigen sterling is prepared by mixing into the calibration object of 3 concentration；
(7) Eu of 1mg the preparation of tracer: is added in 1mg ~ 3mg target antibody3+-N2[P- isocyanic acid-benzyl]-divinyl three
It is mixed in amine tetraacethyl sodium, 4 ~ 35 DEG C of 20 ~ 48h of reaction；Reaction solution uses 50mmol/ through Sephadex G-50 column (1 × 40cm)
L, the Tris-HCl buffer elution of pH7.8, separation tracer conjugate and free Eu3+, use full-automatic fraction collector with 1
The speed of ml/ pipe carries out liquid collection；In carrying out fluorimeter number on fluorescence calculating instrument, collects fluorescence counting in first peak and be greater than
10000000 eluent, 2 ~ 8 DEG C of preservations after mixing；Europium mark working liquid concentration is determined using Checkerboard titration method, then with showing
Track object diluted, then it is lyophilized into microballoon, the microballoon after freeze-drying is transferred in (4) in solid phase reaction cup, seals, retransfers
Into aluminium plastic bag, saved in 2 ~ 8 DEG C；
(8) by above-mentioned redissolution reagent, concentration washing lotion, enhancement solution, solid phase reaction cup, calibration curve card, calibration object, quality-control product and
Tracer packing, labelling, the assembling for completing thyroid function multi-link detection reagent kit.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110286237A (en) * | 2019-06-29 | 2019-09-27 | 山东博科诊断科技有限公司 | Five chemiluminescence detection kits of first function |
| CN113655227A (en) * | 2021-07-02 | 2021-11-16 | 广州俊通生物科技有限公司 | Multi-connected detection kit for screening neonatal diseases, and preparation method and use method thereof |
| CN115541895A (en) * | 2022-11-29 | 2022-12-30 | 天津德祥生物技术股份有限公司 | Formula liquid for improving sensitivity of micro-fluidic inverse detection card and application |
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| CN115541895A (en) * | 2022-11-29 | 2022-12-30 | 天津德祥生物技术股份有限公司 | Formula liquid for improving sensitivity of micro-fluidic inverse detection card and application |
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