CN107045065A - Rapid tuberculosis diagnosis kit and preparation method thereof - Google Patents
Rapid tuberculosis diagnosis kit and preparation method thereof Download PDFInfo
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- CN107045065A CN107045065A CN201710066891.4A CN201710066891A CN107045065A CN 107045065 A CN107045065 A CN 107045065A CN 201710066891 A CN201710066891 A CN 201710066891A CN 107045065 A CN107045065 A CN 107045065A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a kind of rapid tuberculosis diagnosis kit, it includes specific tuberculosis stimulator antigen and test strips, the test strips are sequentially overlapped to be arranged on bottom plate and constituted by sample pad, fluorescence pad, coated film and blotting paper, fluorescent microsphere is combined with the fluorescence pad, fluorescent microsphere coupling, which has, is coated with r interferon monoclonal capture antibody on r interferon binding antibodies, the coated film；The specific tuberculosis stimulator antigen has peptide fragment of 11 amino acid sequences respectively as shown in SEQ ID NO.1 SEQ ID NO.11.
Description
Technical field
The invention belongs to medical science, a kind of rapid tuberculosis diagnosis kit and its preparation side are related particularly to
Method.
Background technology
Current tuberculosis detection technique is divided into four classes：First, Sputum smears, technology is simple, and charge is cheap, is the primary hand of examination
Section, but its positive rate is low, it is impossible to mycobacterium tuberculosis and atylosis mycobacteria are distinguished, its clinical meaning is limited
System；2nd, the immunological detection method based on specific antigen-antibody, such method is simple to operate, but specificity and sensitiveness
It is all bad, it is received limitation in clinical expansion；3rd, the nucleic acid amplification detection method based on Mycobacterium tuberculosis DNA, no matter
Be regular-PCR amplification or isothermal amplification method, its Cleaning Principle be by specific primer detection sample (be usually phlegm
Liquid, hydrothorax, lung lavage) in mycobacterium tuberculosis nucleic acid.The detection method of such PCR-based, susceptibility and specificity are all
Compare high, but need specialty Molecular Laboratory and testing cost is of a relatively high, can only detect pulmonary Mycobacterium, also limit and face
The popularization of bed；4th, the detection kit of tulase IFN-r release is mediated based on cellular immunity, it is more representational at present
It is the QuantiFERON-TB kits that T cell Dot-ELISA (T-SPOT) and Kai Jie companies release, both kits exist
In terms of the sensitivity and specificity of detection, relatively higher level has all been reached, but price is sufficiently expensive, it is difficult to be pushed away as general sieve
Extensively.China is tuberculosis big country, and tuberculosis is also put into national serious infectious diseases ranks, is our High risk populations.Make at present
Tuberculosis screening method, suffers from the defect of different aspect.Therefore, the exploitation of new screening method is to preventing and treating lungy,
And raising China level of medical and health has major and immediate significance.、
The content of the invention
An object of the present invention is to provide a kind of rapid tuberculosis diagnosis kit, and the test strips have high excellent of specificity
Point.
A kind of rapid tuberculosis diagnosis kit, includes specific tuberculosis stimulator antigen and test strips, the test strips by
Sample pad, fluorescence pad, coated film and blotting paper are sequentially overlapped to be arranged on bottom plate and constituted, and fluorescence is combined with the fluorescence pad
Microballoon, fluorescent microsphere coupling, which has to be coated with IFN-r monoclonal on IFN-r binding antibody, the coated film and capture, to be resisted
Body；
The specific tuberculosis stimulator antigen is:Peptide of the amino acid sequence as shown in SEQ ID NO.1-SEQ ID NO.11
Section.
It is a further object to provide a species specificity tuberculosis stimulator antigen.
Concrete technical scheme is as follows.
One species specificity tuberculosis stimulator antigen, chooses Specific Antigen of Mycobacterium Tuberculosis expression region, synthesizes in vitro
Tuberculosis specific polypeptide fragment totally 11.The polypeptide fragment of these synthesis is as the specific stimulator antigen of tuberculosis, for stimulating
Cell to be measured.The specific tuberculosis stimulator antigen has 11 amino acid sequences respectively such as SEQ ID NO.1-SEQ ID NO.11
Shown peptide fragment.
It is a further object to provide a kind of rapid tuberculosis diagnosis test strips.
Concrete technical scheme is as follows.
A kind of rapid tuberculosis diagnosis test strips, the test strips by sample pad, fluorescence pad, coated film and blotting paper sequentially
Overlap joint is arranged on bottom plate and constituted, and fluorescent microsphere is combined with the fluorescence pad, and fluorescent microsphere coupling has IFN-r combination
IFN-r monoclonal capture antibody is coated with antibody, the coated film.
It is a further object to provide the preparation method of above-mentioned rapid tuberculosis diagnosis test strips.
Realize that the technical scheme of above-mentioned purpose is as follows.
The preparation method of above-mentioned rapid tuberculosis diagnosis test strips, comprises the following steps：
IV coated film) is prepared：The IFN-r monoclonal of 0.4-0.6mg/ml concentration is captured into antibody, it is micro- with 0.8-1
Rise nitrocellulose filter per cm and be sprayed onto on nitrocellulose filter dry, slitting, obtain coated film standby；
V fluorescence pad) is prepared：IFN-r binding antibody is coupled on fluorescent microsphere, then the fluorescence for having antibody will be coupled
Microballoon is sprayed on pad, is dried, and slitting obtains fluorescence pad standby；
VI) ready sample pad, pad, coated film and blotting paper are sequentially overlapped and are arranged on bottom plate, institute is produced
State rapid tuberculosis diagnosis test strips.
In one of the embodiments, step II) in, with 90-110ng/ul concentration IFN-r binding antibody, use
The mode that carboxyl-amino covalence is combined is coupled on fluorescent microsphere that (concentration of microballoon is 0.8-1.2mg/ml, fluorescent microsphere and r-
The mass ratio of interferon binding antibody is 9-11：1.
Step II) in, will be coupled has the fluorescent microsphere of antibody to be sprayed on the 6-10 microlitres of amount per cm pads on pad.
The present invention mainly has advantages below：
1) polypeptide antigen of synthesis of the invention is short polypeptide fragment (11-21AA), and its immunogenicity is greatly increased, carried
High stimulation efficiency, and contrast fusion total length antigen or native antigen, cost are relatively low.
2) specific antigen polypeptide, eliminates the influence of BCG vaccine and anonymous mycobacteria, detection specificity is significantly
Improve.
3) special time resolution IFN-r quick determination method, can be completed from detection is loaded onto in 10 minutes,
Time hand-manipulated is only 10 seconds, substantially increases detection efficiency and clinical labor intensity, also turning into high flux examination can
Energy.
Brief description of the drawings
Fig. 1 is the schematic diagram of the rapid tuberculosis diagnosis test strips of the present invention.
Embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In accompanying drawing
Give presently preferred embodiments of the present invention.But, the present invention can be realized in many different forms, however it is not limited to this paper institutes
The embodiment of description.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The implication that technical staff is generally understood that is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply 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, the scope of this specification record is all considered to be.
During one of the present invention implements, it is related to a kind of quick IFN-r detection method (see Fig. 1)：When will contain
Antigen (antibody) of the sample drop of IFN-γ in sample application zone, testing sample is anti-with the fluorescent nanometer microsphere mark in pad
Body merga pass capillarity is chromatographed forward, after detection zone is reached, is closed with antibody fixed in detection line, formation particulate-anti-
Body-Ag-Ab sandwich complex is simultaneously fixed in detection line, and unnecessary Fluorescent microsphere marker continues to chromatograph forward,
With being fixed on the anti-binding of nature controlling line two.After reaction terminates, with ultraviolet source (365nm) to detection zone Scanning Detction, detection line and
Fluorescent nanometer microsphere sends the fluorescence (615nm) of high intensity on nature controlling line, and decay time is also longer.During using delaying measurement
Between, after abiogenous short life fluorescence all decays in sample substrate, then the specificity fluorescent of microballoon is measured, thus may be used
To exclude the interference of special background fluorescence completely.Pass through detection line and the power and its ratio of nature controlling line fluorescence intensity, you can point
Separate out the concentration of determinand in sample.
In the present embodiment, the rapid tuberculosis diagnosis kit rapid tuberculosis diagnosis kit includes specific tuberculosis
Stimulator antigen and test strips, its preparation method and detection are as described below.
1) specific tuberculosis stimulator antigen polypeptide fragment:TEQQWNFAGIEAAASA(SEQ ID NO.1)；
IEAAASAIQGNVTS(SEQ ID NO.2)；AIQGNVTSIHSLLDEGKQSLTKL(SEQ ID NO.3)；
QSLTKLAAAWGGSGSEAYQGVQ(SEQ ID NO.4)；GSEAYQGVQQKWDATATELNNA(SEQ ID NO.5)；
NFERISGDLKTQIDQVESTAGSL(SEQ ID NO.6)；QVESTAGSLQGQWRGAAGTMQ(SEQ ID NO.7)；
RQAGVQYSRAD E EQQQALSS(SEQ ID NO.8)；LFAA TAAAAAA VDRGDPP(SEQ ID NO.9)；
ASAAALAGDAAGAWR(SEQ ID NO.10)；MSGHALAARTLLAAA(SEQ ID NO.11).
2) polypeptide of above-mentioned fragment is synthesized first, and purity need to be more than 85%.Secondly, by 11 kinds of polypeptide fragments etc. than mixing
Afterwards, DMSO (dimethyl sulfoxide (DMSO)) dissolvings (adding 100ulDMSO per 50mg polypeptides) are first added, then with the DPBS of endotoxin-free
(Du Shi phosphate buffers) is diluted to 5mg/ml solution for standby, be stored in -20 degrees Celsius it is standby.Used time taking heparin lithium anti-freezing
Whole blood 1ml, adds above-mentioned antigen mixed liquor 5ul, in 37 C overnight cultures, separated plasma is to be checked.
3) whole blood cells stimulation test：The heparin tube of heparin lithium anti-freezing on probation, takes at least 3.5ml blood, and examination is overturned immediately
Pipe is mixed several times.Three blank heparin tubes are taken, 5ul PBS solutions are separately added into, labeled as negative control pipe (N), 5ul concentration
For 5mg/ml PHA solution, labeled as positive control pipe (P), 5ul concentration is 5mg/ml above-mentioned antigenic solution.Per Guan Zhongjia
Enter 1ml blood samples, and in 37 degrees Celsius, cultivate 16-24 hours.Whole blood after culture is centrifuged 15 minutes in 3000g, separated plasma,
It is to be measured.
4) a kind of preparation of IFN-r dry type fluorescence immunoassay test strips：A) raw material prepare：Nitrocellulose filter (NC
Film) coating, IFN-r monoclonal is captured into antibody and (opens Thailand, clone number in Hangzhou：MJH01), according to those skilled in the art's
Conventional practices, with 0.5mg/ml concentration, are sprayed onto on film with 0.9 microlitre of nitrocellulose filter per cm, and Celsius 37
Degree is dried 12 hours, is cut standby (2.5cm x 30cm)；B) raw material prepare：The processing of fluorescence pad, IFN-r is combined anti-
Body (opens Thailand, clone number in Hangzhou：MJH03) with 100ng/ul concentration, the mode combined using carboxyl-amino covalence is coupled to dense
Spend on the fluorescent microsphere for 1mg/ml, the amount ratio of fluorescent microsphere and binding antibody is 10：1 (mass ratio)；Coupling is had anti-again
The fluorescent microsphere of body is sprayed on pad (glass fibre) with every 8 microlitres of cm pads with 1mg/ml concentration and is obtained fluorescence pad,
And dried 12 hours in 37 degrees Celsius, cut standby (1cm x 30cm).Antibody, the concentration of microballoon and consumption used above,
Constant according to total amount used, the general knowledge that those skilled in the art prepares according to test strips correspondingly can convert and change.
C) prepared by sample pad：Shanghai gold target glass, by 0.01M PB solution, (sodium dihydrogen phosphate+disodium hydrogen phosphate delays
Fliud flushing, pH7.4) immersion, then dry 12 hours, cut standby (1.5cm x 30cm) in 37 degrees Celsius.
D) holder prepares：Blotting paper (3.1cm x 30cm) and PVC (8cm x 30cm) plate are cut into dimension, it is standby
With.
E) by the raw material of above-mentioned pretreatment according to sample pad (sample pad is glass fibre), pad, coated film, water suction
The order of paper, is attached on bottom plate (PVC board), there is the overlapping of 1 millimeter between each component.
F) cut：The big plate posted is cut into the test strips of 4mm X 8cm specifications, and is fitted into neck, finished product is packaged into,
Be put in storage after quality inspection standby.The test strips are obtained, the test strips are sequentially taken by sample pad, fluorescence pad, coated film and blotting paper
Connect to be arranged on bottom plate and constitute, be combined with fluorescent microsphere on the fluorescence pad, it is anti-that fluorescent microsphere coupling has IFN-r to combine
IFN-r monoclonal capture antibody is coated with body, the coated film.
5) IFN-r test strip is taken, 50ul test plasmas are added, reacted 10 minutes, dry type immunofluorescence point is utilized
Analyzer measures fluorescent value, and is converted into IFN-r concentration (Shi Douhui that dispatched from the factory per a batch of test strips is with fluorescent value
The standard curve of correspondence interferon concentration, standard curve be by 0.25IU, 1IU, 4IU, what 8IU standard items were quantitatively got).
Each sample does negative control and positive control and stimulated simultaneously.As a result interpretation such as following table：Negative control (N), positive control
(P), antigenic stimulus (T)；Unit IU/ML.
Embodiment 2
First, testing goal
The present embodiment uses rapid tuberculosis diagnosis kit and detection method described in embodiment 1, evaluates the described kit
Detection results, and corresponding specificity and sensitivity.
2nd, sample is detected
Detection sample in table 1 passes through for the clinical sample that Guangdong Provincial No.2 People's Hospital's (Guangdong Province's Contingency Hospital) undertakes
Laboratory Diagnosed method confirms as the tuberculosis positive and each 78 of negative sample.
Detection sample in table 2 undertakes clinical sample by real for Guangdong Provincial No.2 People's Hospital's (Guangdong Province's Contingency Hospital)
Test the tuberculosis positive sample 60 that room diagnosis method confirms as BCG vaccination person, atypical mycobacterial infection person 22.
(Guangdong Provincial No.2 People's Hospital (Guangdong Province should for the whole blood sample that sample is 219 clinical acquisitions of detecting in table 3
Anxious hospital).The detection of the IFN-r of contrast agent uses ELISA detection modes, and operation is completed according to reagent specification
Carry out.
3rd, experimentation：
1) whole blood sample is gathered：Using the heparin tube of heparin lithium anti-freezing, whole blood sample is gathered, each sample size is no less than
After 3.5ml. collection blood, heparin tube being overturned immediately, is mixed, the blood sample of collection is no more than 4 hours in room temperature preservation, applied to anti-
Primary stimuli is tested.
2) antigenic stimulus is tested：Three blank heparin tubes are taken, P (positive control pipe), N (negative control pipe), T are marked respectively
(tuberculosis antigen stimulates pipe).By the whole blood of collection, draw 1ml and be separately added into three corresponding pipes.5ul is added in P pipes
Concentration is former (being purchased from Sigma) for 10mg/ml lectins；The PBS solution that 5ul is added in N pipes (is not added with tuberculosis antigen thorn
Swash liquid)；5ul concentration is added in T pipes stimulates liquid for 10mg/ml above-mentioned tuberculosis antigen；After sample-adding, heparin tube is overturned some
It is secondary, to mix.Then, 37 degree of baking ovens, incubated overnight (- 24 hours 16 hours) are stood.
3) IFN-r concentration mensuration：By the ischemic of incubated overnight, centrifuged 15 minutes in 3000g, take supernatant to be used to detect.
50ul supernatants are taken, IFN-r quantitative testing test paper bar is added, reacted 15 minutes, using fluorescence immunity analyzer, r- are read
The concentration of interferon.Result judgement mode is shown in above table.
4th, testing result
Table one：Yin and yang attribute coincidence rate
Table two：Specificity experiments
| Sample type | Clinical diagnosis | Embodiment result | Specificity |
| Whole blood sample | 60 tuberculosis feminine genders, BCG vaccination person | 60 feminine genders | It is good |
| Whole blood sample | 22 tuberculosis feminine genders, atypical mycobacterial infection person | 22 feminine genders | It is good |
Table three：The experiment of comparison property
Sensitivity：121/ (121+2) x 100%=98.4%
Specificity：95/ (95+1) x 100%=99.0%
Total coincidence rate：(121+95)/(121+1+2+95) x 100%=98.6%.
Interpretation of result：The whole blood sample of 219 clinical acquisitions, is respectively used to stimulate altogether.The blood plasma of collection, is disturbed with r-
The method detection of plain quantitative testing test paper bar or ELISA.Our result show that, detection kit is surveyed altogether described in embodiment
122 positives, 97 negative samples.And 123 positives of reagent survey are compared, 96 feminine genders.Detection kit described in embodiment 1
Sensitivity be 98.4%, specificity be 99%, with very high sensitivity and specificity.Embodiment and comparison reagent are (triumphant
The QFT products of outstanding company) total coincidence rate be 98.6%, with very high uniformity.
It should be noted that specific tuberculosis stimulator antigen polypeptide fragment described in detection kit described in embodiment 1, should
Above-mentioned 11 kinds should be included while using, inventor has found in an experiment, this 11 polypeptides, if lacking wherein one, positive rate
It will decline, by statistical analysis, find wherein to lack one, positive rate would generally will decline 15-25%.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
SEQUENCE LISTING
<110>Guangzhou Bao Chuan Bioisystech Co., Ltd
<120>Rapid tuberculosis diagnosis kit and preparation method thereof
<160> 11
<170> PatentIn version 3.3
<210> 1
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<213>Artificial sequence
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Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala
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Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser
1 5 10
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Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
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Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu
1 5 10 15
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Gly Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala
1 5 10 15
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Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val
1 5 10 15
Glu Ser Thr Ala Gly Ser Leu
20
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Gln Val Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala
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1 5 10 15
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Claims (9)
1. a kind of rapid tuberculosis diagnosis kit, it is characterised in that include specific tuberculosis stimulator antigen and test strips, described
Test strips are sequentially overlapped to be arranged on bottom plate and constituted by sample pad, fluorescence pad, coated film and blotting paper, are tied on the fluorescence pad
Conjunction has fluorescent microsphere, and fluorescent microsphere coupling has is coated with IFN-r Dan Ke on IFN-r binding antibody, the coated film
Grand capture antibody；
The specific tuberculosis stimulator antigen has 11 amino acid sequences respectively as shown in SEQ ID NO.1-SEQ ID NO.11
Peptide fragment.
2. rapid tuberculosis diagnosis kit according to claim 1, it is characterised in that coated film in the test strips
Preparation method is：The IFN-r monoclonal of 0.4-0.6mg/ml concentration is captured into antibody, with 0.8-1 microlitres of nitric acid per cm
Cellulose membrane is sprayed onto on nitrocellulose filter and dried, and slitting obtains coated film.
3. rapid tuberculosis diagnosis kit according to claim 1 or 2, it is characterised in that the fluorescence in the test strips
The preparation method of pad is：The mode that IFN-r binding antibody application carboxyl-amino covalence is combined is coupled on fluorescent microsphere,
The concentration of the IFN-r binding antibody is 90-110ng/ul, and the concentration of fluorescent microsphere is 0.8-1.2mg/ml, fluorescent microsphere
Mass ratio with IFN-r binding antibody is 9-11：1；
To be coupled has the fluorescent microsphere of antibody to be sprayed on the 6-10 microlitres of amount per cm pads on pad, produces.
4. a species specificity tuberculosis stimulator antigen, it is characterised in that it is amino acid sequence respectively such as SEQ ID NO.1-SEQ
Peptide fragment shown in ID NO.11.
5. a kind of rapid tuberculosis diagnosis test strips, it is characterised in that the test strips are by sample pad, fluorescence pad, coated film and inhale
Water paper is sequentially overlapped to be arranged on bottom plate and constituted, and fluorescent microsphere is combined with the fluorescence pad, and fluorescent microsphere coupling has r- to do
Disturb and IFN-r monoclonal capture antibody is coated with plain binding antibody, the coated film.
6. the preparation method of rapid tuberculosis diagnosis test strips described in claim 5, it is characterised in that comprise the following steps：
I coated film) is prepared：The IFN-r monoclonal of 0.4-0.6mg/ml concentration is captured into antibody, with 0.8-1 microlitres every li
Rice nitrocellulose filter is sprayed onto on nitrocellulose filter and dried, and slitting obtains coated film, standby；
II fluorescence pad) is prepared：IFN-r binding antibody is coupled on fluorescent microsphere, then the fluorescent microsphere for having antibody will be coupled
It is sprayed on pad, dries, slitting obtains fluorescence pad standby；
III) ready sample pad, fluorescence pad, coated film and blotting paper are sequentially overlapped and is arranged on bottom plate, is produced described
Rapid tuberculosis diagnosis test strips.
7. preparation method according to claim 6, it is characterised in that step II) in, by 90-110ng/ul concentration r-
Interferon binding antibody, the mode combined using carboxyl-amino covalence is coupled on fluorescent microsphere, the concentration of the fluorescent microsphere
For 0.8-1.2mg/ml, the mass ratio of fluorescent microsphere and IFN-r binding antibody is 9-11：1；Then coupling there is into antibody
Fluorescent microsphere is sprayed on pad with the 6-10 microlitres of amount per cm pads, is produced.
8. the preparation method according to claim 6 or 7, it is characterised in that, by the IFN-r list of 0.5mg/ml concentration
Clone's capture antibody, is sprayed onto on nitrocellulose filter with 0.9 microlitre of nitrocellulose filter per cm and dried, and slitting is coated with
Film.
9. preparation method according to claim 7, it is characterised in that step II) in, 100ng/ul concentration r- is disturbed
Plain binding antibody, the mode combined using carboxyl-amino covalence is coupled on fluorescent microsphere, and the concentration of fluorescent microsphere is 1mg/
The mass ratio of ml, fluorescent microsphere and IFN-r binding antibody is 10：1；Then coupling there is into the fluorescent microsphere of antibody with 8 microlitres
It is sprayed on per the amount of cm pads on pad, produces the fluorescence pad.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020111393A1 (en) * | 2018-11-27 | 2020-06-04 | 바디텍메드(주) | Tuberculosis diagnosis method and apparatus therefor |
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