CN101303355A - Tuberculosis special antigen rapid diagnosis kit - Google Patents
Tuberculosis special antigen rapid diagnosis kit Download PDFInfo
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- CN101303355A CN101303355A CNA2007100543756A CN200710054375A CN101303355A CN 101303355 A CN101303355 A CN 101303355A CN A2007100543756 A CNA2007100543756 A CN A2007100543756A CN 200710054375 A CN200710054375 A CN 200710054375A CN 101303355 A CN101303355 A CN 101303355A
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- tuberculosis
- monoclonal antibody
- desa
- specific antigen
- rapid diagnosis
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Abstract
The invention utilizes a genetic engineering method to express in vitro the desA protein of mycobacterium tuberculosis and produces monoclonal antibodies by using the protein. A tuberculosis specific antigen rapid diagnosis reagent kit is produced by utilizing enzyme linked immunization, chemiluminescence, colloidal gold test strip method and other diagnostics methods so as to detect MTB quickly with low cost. The technique has wide application value in clinical diagnosis.
Description
Technical field
The invention belongs to the immunodiagnosis field, be specifically related to a kind of tuberculosis special antigen rapid diagnosis kit.
Background technology
Tuberculosis is worldwide important economy and medical problem.Nineteen ninety-five dies from number lungy in the world and surpasses any infectious diseases.It is 55% in untreated crowd that case mortality is estimated, is 15% in the patient of treatment.As a kind of ancient infectious disease, epidemic situation lungy is in rising trend in the whole world.Estimate that according to WTO the whole world has 2,000,000,000 people to infect tubercle bacillus approximately at present, and 8,000,000 new cases and 3,000,000 people's death [1] is arranged every year.In China, according to 2000 the 4th time national tuberculosis survey estimates, the whole nation has 5.5 hundred million tuberculosis cases approximately, nearly half of national population [2].The present form sternness lungy of China.Setting up advanced diagnostic techniques and treatment measure as early as possible, is a urgent task.Not only being related to ten million people's body and mind health, also is the major issue that is related to national economy and the people's livelihood and national economic development.
Along with the progress of diagnostic techniques and to deepening continuously that Much's bacillus is studied, the experimental technique of diagnosis of tuberculosis emerges in an endless stream, and emerges many new methods of inspection [3].At present, bacteriological analysis is still the diagnosis of tuberculosis common method.The phlegm smear for microscopic examination is the foundation of making a definite diagnosis, and also is to find that tuberculosis is the most classical, the most effective means.For recall rate is provided, also can use fluorescence microscopy.Serological method is lungy quick, and accurately diagnosis provides another kind of selection.Since the antigenicity of Much's bacillus a little less than, common antigen decision family is arranged between genus and between planting, humoral immunity and correlativity lungy are not too definite, thereby seek sensitivity and all high monomeric protein of specificity is a focus of present this area research.In addition, PCR has the susceptibility height, high specificity and advantage such as quick, but can not differentiate the life or death of infectious bacteria, simultaneously also owing to the very easily contaminated false positive results that produces, bring uncertainty to diagnosis.PCR-fluorogenic probe hybridzation method, PCR-micropore plate hybridization method, PCR-molecular beacon method etc. all has easy, fast, antipollution, highly sensitive, advantages such as high specificity belong to more advanced diagnosis of molecular biology technology, also are the effective ways of tuberculosis auxiliary diagnosis.Biochip technology is the technology that newly-developed gets up, and is mainly used in Mycobacterium tuberculosis drug-resistant gene mutation check, has quick and sensitive advantage.Though ligase chain reaction does not have the highly sensitive of PCR, have than higher recognition capability, be applicable to the tuberculosis primary dcreening operation, can be used for early diagnosis.Characteristics such as Much's bacillus bacteriophage method has cheapness, and is simple, special can be differentiated the life or death of bacterium to be expected to become the chemical sproof Fast Detection Technique of tulase simultaneously.Yet these methods all have certain characteristics and advantage at present, but have some defectives in various degree.Therefore, finding and set up more perfect and accurate method of diagnosis of tuberculosis efficiently or technology is not only necessary, but also quite urgent for diagnosis lungy and treatment.
In all above-mentioned diagnostic techniquess, serological technique mainly is based on chromogenic reaction or the enzyme-catalyzed chemical luminescence reaction or the colloidal gold-labeled method of classical antigen-antibody reaction and enzyme connection, and is reliable because of it, easy and favored fast.Behind the organism infection tulase, can produce various antibody, inducing the antigen that produces these antibody is the various components of Much's bacillus, and wherein the important composition of a class is each albuminoid (enzyme) of this bacterium secretion.Found more than 100 kind of secretory protein in the culturing filtrate of Much's bacillus, these albumen play an important role in the immune response of tuberculosis patient, and therefore good utilization prospect is arranged in diagnosis lungy and prevention and treatment thereof.Seek the strong and high tuberculosis antigen of kind specificity of new immunogenicity, the diagnostic method that development is quick, responsive, special is timely discovery, treatment and control key lungy.
Summary of the invention
The present invention is intended to by gene engineering expression and purifying desA albumen, makes the desA monoclonal antibody, the method that provides a kind of tuberculosis serological to detect.Its concrete application process comprises the enzyme linked immunoassay method, enzyme-catalyzed chemical luminescence method and Collaurum marking etc.
Euzymelinked immunosorbent assay (ELISA) (ELISA): utilize double antibody sandwich method that one strain desA monoclonal antibody solid phase is marked on little reacting hole in enzyme, with another strain desA monoclonal antibody or how anti-mark horseradish peroxidase (HRP), by the tuberculosis specific antigen in the TMB color developing detection serum.The TMB chromogenic substrate is replaced by the Luminol chemical luminous substrate, concrete content that can the detection by quantitative tuberculosis antigen.Its advantage is that detection signal is strong, and the range of linearity is wide.With colloid gold label DesA monoclonal antibody or how anti-, utilize immunologic paper to analyse the collaurum fast diagnose test paper bar that technological development goes out in addition, have highly sensitively, quick and convenient, do not need with advantages such as expensive detection equipment.
Embodiment
Embodiment 1, euzymelinked immunosorbent assay (ELISA) (ELISA) detect the tuberculosis specific antigen.
Adopt gene engineering expression and purifying desA albumen and make desA monoclonal antibody bag by elisa plate, with another strain monoclonal antibody or how anti-mark horseradish peroxidase, form kit with other reagent, use the tuberculosis specific antigen in double antibody sandwich method detection human serum or the blood plasma, be used for the examination that clinical tuberculosis patient infects.
[kit component]
(1) surveys tuberculosis 1 of elisa plate of specific antigen (96 hole)
(2) 1 bottle of sample diluting liquid (7ml)
(3) survey tuberculosis 1 bottle of enzyme labelled antibody of specific antigen (13ml)
(4) positive control (deactivation) 1 bottle (1ml)
(5) negative control (deactivation) 1 bottle (1ml)
1 bottle of (6) 20 * cleansing solution (50ml)
(7) 1 bottle of developer A (7ml)
(8) 1 bottle of developer B (7ml)
(9) 1 bottle of stop buffer (7ml)
(10) the adhesive sticker mounting is 2
(11) instructions is 1 part
(12) sealing bag, drying agent 1 cover
[sample requirement]
Serum or blood plasma (available sodium citrate, heparin etc. are made anti-coagulants), strictness is treated clinical samples by tuberculosis infection sources safeguard procedures.
[trace routine]
(1) all ingredients is moved on to room temperature (18-25 ℃) balance after half an hour, get one bottle of 20 * cleansing solution, adding distil water is to 1000ml, and is standby behind the mixing.
(2) elisa plate is taken out from sealing bag, every hole adds 50 μ l sample diluting liquids, establishes a blank hole, two positive control holes, two negative control holes.Each 50 μ l adds in control wells after getting the abundant mixing of positive control and negative control difference, the blank hole only adds the sample dilution, and all the other each holes add serum 50 μ l to be checked, fully mixing, enzyme-linked reaction plate is put mixing 5-10 second on the microplate oscillator, with adhesive sticker mounting capping reaction plate.Unspent lath is preserved in sealing bag.
(3) elisa plate was put 37 ℃ of incubations 30 minutes, carefully blotted serum in the hole, washed 5 times with cleansing solution, patted dry on thieving paper then.
(4) every hole adds enzyme labelled antibody 100 μ l except that blank well, with adhesive sticker sheet capping elisa plate.37 ℃ of incubations 30 minutes are washed plate 5 times, pat dry.
(5) every hole adds developer A 50 μ l, developer B 50 μ l, and vibration rearmounted 37 ℃ of dark places colour developing is 10 minutes gently, and every hole adds stop buffer 50 μ l.
(6) select microplate reader to measure wavelength 450nm, use the blank well zeroising, measure each hole A value.Also can select 630nm as the reference wavelength, measure with dual wavelength.
[result's judgement]
(1) Cutoff value=0.10+N (mean value of negative control)
(2) if, pressing 0.02 less than 0.02 o'clock, negative control A value calculates.
(3) if positive control A value less than 0.5, negative control A value is greater than 0.1, then experiment is false, the whole experiment reformed.
(4) sample A value is negative less than the Cutoff value, and is positive more than or equal to the Cutoff value.
(5) the positive person diplopore retrial of must resampling is confirmed.
[points for attention]
(1) applying unit of this reagent must be the tuberculosis primary dcreening operation laboratory of local administrative department of public health approval.
(2) all testing must meet tuberculosis experiment management standard and bio-safety rules regulation, and strictness prevents cross-infection.Must wear gloves during operation, wear work clothes, strict sound and execution disinfection and isolation system.
(3) judgement of testing result must be as the criterion with the microplate reader reading.
(4) sample and dilution are all used the charger filling, and often proofread accuracy.
(5) all samples, cleansing solution and various refuse, gurry all should be handled by infective agent.
(6) each batch reagent can not be used with.
When (7) microwell plate took out from cold storage environment, balance was done to moisture and can be used to the greatest extent at room temperature, and the person of not using up need put into the sealing bag of drying agent and preserve.
(8) sample that is used to detect should keep fresh.
(9) remaining sample and discarded object should be through 121 ℃ of autoclavings 30 minutes, or handle with sanitizers such as sodium hypochlorite and to abandon it after 30 minutes.
Embodiment 2, chemoluminescence method detect examination tuberculosis specific antigen
Principle and purposes: adopt gene engineering expression and purifying desA albumen and make desA monoclonal antibody bag by the luminous microwell plate of opaque chemistry, with another strain monoclonal antibody or how anti-mark horseradish peroxidase, use the tuberculosis specific antigen in double antibody sandwich method detection human serum or the blood plasma, last enzyme-catalyzed chemical luminescence substrate amplifies detection signal, tested antigenic content is directly proportional with the luminous signal power, thereby reaches the purpose of detection by quantitative.Be used for the examination that clinical tuberculosis patient infects.
Kit is formed:
| Numbering | Title | Quantity | Explanation |
| 1 2 3 4 5 6 7 8 | Bag is by plate enzyme conjugates standard items luminous substrate A luminous substrate B solid washing lotion envelope film instructions | 11 part of 1 bottle 51 bottle 1 bottle 2 bag in 96 holes | 8 holes * 12 a 6ml S1, S2, S3, S4, S5 3.5ml 3.5ml uses the 500ml dissolved in distilled water respectively |
Collection of specimens: serum or blood plasma (available sodium citrate, heparin etc. are made anti-coagulants), strictness is treated clinical samples by tuberculosis infection sources safeguard procedures.
Points for attention:
Kit only donor is diagnosed use outward.Read over operation instructions before the operation, carry out test operation, to guarantee reliable results, good reproducibility in strict accordance with instructions in the kit.
(1) applying unit of this reagent must be the tuberculosis primary dcreening operation laboratory of local administrative department of public health approval.
(2) all testing must meet tuberculosis experiment management standard and bio-safety rules regulation, and strictness prevents cross-infection.Must wear gloves during operation, wear work clothes, strict sound and execution disinfection and isolation system.Need wear disposable glove when reagent treatment and sample, should wash hands thoroughly after the operation.All samples should be considered as potential infectious substance, during waste treatment, please carry out according to local government and the countries concerned's regulation.
(3) after bag is opened by bar, the residue bag should be covered with adhesive sticker envelope film by bar, sealing is preserved in the bag of packing into, in order to avoid make moist.
(4) feed head can not be used with, in order to avoid cross pollution.
(5) strict per reaction time in step of control and temperature, operation should be compact.
(6) washing is wanted thoroughly, and washing lotion should be filled with every hole, but unavailable water is too quickly, avoids producing bubble.Each washing all should dry liquid in the hole, liquid in the hole should be patted dry at last.Recommend to use the plate machine of washing.
(7) reagent please uses before the deadline.To in time be placed under the 2-8 ℃ of condition after kit uses and store.
(8) for guaranteeing result's accuracy, should under room temperature (18-25 ℃) condition, operate.
Operation steps:
* prepare before the experiment
(1) guarantees operating environment at room temperature (18-25 ℃), take out kit and testing sample.All reagent and sample are returned to room temperature (needing 30 minutes approximately).
(2) constant temperature oven or water-bath are transferred to temperature of reaction.
(3) the liquid standard product can directly use; The solid standard items are fully dissolving back use (suggestion dissolving 5 minutes) on request.
(4) the configuration washing lotion is about to every bag solid washing lotion and is dissolved in 500ml distilled water or deionized water.
(5) before the experiment, it is volume required that luminous substrate A, B liquid equal proportion are mixed to this test.* experimental procedure
(1) takes out a certain amount of bag by the hole, numbering.Add 50 μ l standard items (according to B0, S1, S2, S3, S4 order) and blood serum sample 50 μ l by the hole.
(2) every hole adds enzyme conjugates 50 μ l respectively, and vibration mixed it in 60 seconds on the micro oscillator.
(3) paste upper sealing film, put 37 ℃ of incubations 60 minutes.
(4) get rid of potpourri in the hole,, on thieving paper, pat dry at last with washing plate machine washing plate 6 times.
(5) every hole adds mixed substrate 50 μ l, and the reaction of room temperature (18-25 ℃) lucifuge was measured in 15 minutes.
(6) detect luminous intensity with chemiluminescence detector.
The result calculates:
This kit recommend to adopt point-to-point to fit mode, and promptly the logarithm value with the standard items concentration value is horizontal ordinate (X-axis), is ordinate (Y-axis) with the logarithm value of standard items luminous intensity values, sets up typical curve, calculates.
Advise that each laboratory sets up range of normal value according to own actual conditions and exposed population group.This kit is only made one of supplementary means of diagnosing, for clinician's reference.
Kit is stored: this kit should be in 2-8 ℃ of storage, the term of validity six months.
Embodiment 3, immune colloid gold method detect the tuberculosis specific antigen
Principle and purposes: diagnostic method lungy has looks into acid-fast bacilli, phlegm (or body fluid) cultivation, tuberculin experiment and radiograph test in the phlegm.And these methods do not reach required diagnosis susceptibility.Being used for best index clinical and stream accent diagnosis of tuberculosis is the serology experiment, such as blood clotting method, complement fixation test, immunofluorescence technique and radioimmunodiffusion technology.But these methods need expensive equipment and instrument material and have been subjected to serious restriction in non-developed country.
The present invention uses the tuberculosis specific antigen in double antibody sandwich method detection human serum or the blood plasma, adopt gene engineering expression and purifying desA albumen and make desA monoclonal antibody bag by nitrocellulose filter, with another strain monoclonal antibody of colloid gold label or many anti-evenly coating on the all-glass paper, the chromogenic reaction that the utilization ply of paper is analysed technology and immunity gold reaches the purpose that detects the tuberculosis specific antigen.Be used for the examination that clinical tuberculosis patient infects.
Specification: 20 person-portions/box
Product is formed: tuberculosis antigen test card: 1 part in 20T instructions
Collection of specimens: serum or blood plasma (available sodium citrate, heparin etc. are made anti-coagulants), strictness is treated clinical samples by tuberculosis infection sources safeguard procedures.
Experimental procedure:
(1) reagent, sample balance are to room temperature.
(2) take out test card, and on card, indicate patient name or numbering.
(3) in the sample well of reaction card, add 40 μ l serum (or 60 μ l whole bloods).
(4) in the sample well of reaction card, accurately add 5 dilutions (200 μ l).
(5) when proceeding to 5 minutes, reaction in sample well, adds 1 dilution again.
(6) react 15-20 minute sentence read result again.
Interpretation as a result:
(1) feminine gender: have only 1 pink/purple band (nature controlling line).
(2) positive: 2 pink/purple bands (nature controlling line and detection line) occur.
(3) invalid: if there is not tangible band to appear at reaction zone and Quality Control district, it is invalid to test.The suggestion repeated experiments.
Points for attention:
(1) applying unit of this reagent must be the tuberculosis primary dcreening operation laboratory of local administrative department of public health approval.
(2) all testing must meet tuberculosis experiment management standard and bio-safety rules regulation, and strictness prevents cross-infection.Must wear gloves during operation, wear work clothes, strict sound and execution disinfection and isolation system.Need wear disposable glove when reagent treatment and sample, should wash hands thoroughly after the operation.All samples should be considered as potential infectious substance, during waste treatment, please carry out according to local government and the countries concerned's regulation.
(3) leave the kit of refrigerator in, fully recover the use of unpacking again after the room temperature.
(4) please in the kit term of validity, use.
Storage requirement and effect phase: 4～30 ℃ of dry places of shady and cool lucifuge store, and the term of validity is 18 months.
Claims (3)
1, tuberculosis special antigen rapid diagnosis kit, it is characterized in that, mainly formed by elisa plate, tuberculosis specific antigen enzyme labelled antibody and other reagent by tubercle bacillus secretory protein desA monoclonal antibody bag, use the tuberculosis specific antigen in double antibody sandwich method detection human serum or the blood plasma, be used for the examination that clinical tuberculosis patient infects.
2, tuberculosis special antigen rapid diagnosis kit, it is characterized in that, mainly formed by the luminous microwell plate of opaque chemistry, monoclonal antibody or how anti-mark horseradish peroxidase and other reagent by tubercle bacillus secretory protein desA monoclonal antibody bag, use the tuberculosis specific antigen in double antibody sandwich method detection human serum or the blood plasma, last enzyme-catalyzed chemical luminescence substrate amplifies detection signal realizes detection by quantitative, is used for the examination that clinical tuberculosis patient infects.
3, tuberculosis special antigen rapid diagnosis kit, it is characterized in that, with tubercle bacillus secretory protein desA monoclonal antibody bag by nitrocellulose filter, with another strain monoclonal antibody of colloid gold label or many anti-evenly coat on the all-glass paper, use ply of paper and analyse the chromogenic reaction detection tuberculosis specific antigen of technology and immunity gold, be used for the examination that clinical tuberculosis patient infects.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100543756A CN101303355A (en) | 2007-05-10 | 2007-05-10 | Tuberculosis special antigen rapid diagnosis kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100543756A CN101303355A (en) | 2007-05-10 | 2007-05-10 | Tuberculosis special antigen rapid diagnosis kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102246040A (en) * | 2008-12-15 | 2011-11-16 | 开普敦大学 | Method and device for diagnosing tuberculosis |
| CN102426244A (en) * | 2011-11-21 | 2012-04-25 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN102660559A (en) * | 2012-04-28 | 2012-09-12 | 吉林大学 | Mycobacterium tuberculosis (TB) recombinant protein and preparation method thereof |
| CN105486860A (en) * | 2014-10-09 | 2016-04-13 | 中国人民解放军军事医学科学院基础医学研究所 | Mycobacterium tuberculosis antigen detection method based on specific multi-antibody |
| CN111443198A (en) * | 2020-03-19 | 2020-07-24 | 济南杏恩生物科技有限公司 | Method for rapidly detecting tubercle bacillus secretory protein based on colloidal gold method |
-
2007
- 2007-05-10 CN CNA2007100543756A patent/CN101303355A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102246040A (en) * | 2008-12-15 | 2011-11-16 | 开普敦大学 | Method and device for diagnosing tuberculosis |
| CN102246040B (en) * | 2008-12-15 | 2014-11-05 | 安特鲁姆生物技术（私人）有限公司 | Method and device for diagnosing tuberculosis |
| CN102426244A (en) * | 2011-11-21 | 2012-04-25 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN102426244B (en) * | 2011-11-21 | 2014-04-09 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN102660559A (en) * | 2012-04-28 | 2012-09-12 | 吉林大学 | Mycobacterium tuberculosis (TB) recombinant protein and preparation method thereof |
| CN105486860A (en) * | 2014-10-09 | 2016-04-13 | 中国人民解放军军事医学科学院基础医学研究所 | Mycobacterium tuberculosis antigen detection method based on specific multi-antibody |
| CN111443198A (en) * | 2020-03-19 | 2020-07-24 | 济南杏恩生物科技有限公司 | Method for rapidly detecting tubercle bacillus secretory protein based on colloidal gold method |
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Application publication date: 20081112 |