CN101762692A - Magnetic immunochromatographic strip for detection of tuberculosis (TB) antibody in blood and preparation method thereof - Google Patents
Magnetic immunochromatographic strip for detection of tuberculosis (TB) antibody in blood and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a magnetic immunochromatographic strip for detection of tuberculosis (TB) antibodies in blood and a preparation method thereof. The strip is prepared through the following steps: pasting a coating film, a magnetic particle pad combined with TB antigens, a sample pad and an absorbent pad on a bottom board in sequence at intervals of 2mm, and using a transparent plastic seal film to cover the upper layer. The coating film is pre-coated with TB antigen detection lines and quality control lines. The invention introduces the magnetic immunochromatographic technique into the detection of the TB antibody, thereby greatly improving the detection sensitivity and accuracy, shortening the detection window period and reducing the labor intensity of the operating personnel.
Description
Technical field
The present invention relates to the magnetic immuno-chromatographic test paper strip and preparation method thereof of a kind of TB of detection antibody of field of medical examination.
Background technology
(Tuberculosis is common and a kind of infectious disease that can be fatal TB) to tuberculosis, is caused by mycobacterium (mainly be Much's bacillus [Mycobacterium tuberculosis], claim " tubercle bacillus " [tubercle bacillus] again).Tuberculosis infects usually and destroys lung and (claims " pulmonary tuberculosis ", claim " tuberculosis " again) and lymphatic system (title " tuberculosis lymph pathology ", claim " scrofula " again), but other organ such as brain, central nervous system, the circulation system, urinary system, bone, joint even skin also can infected (can cause " tubercular meningitis " as infecting brain).Other mycobacterium, also can cause tuberculosis, but not infect health adult usually as Mycobacterium bovis (Mycobacterium bovis), mycobacterium africanum (Mycobacteriumafricanum), Ka Shi mycobacterium (Mycobacterium canetti), mycobacterium microti (Mycobacteriummicroti).
Existing 600,000,000 people in the whole world, promptly 1/10th of global population, infecting has tubercle bacillus.Most sufferer does not have illness, be called the tuberculosis infection of hiding (latent TB infection), but wherein the latent infection person of about 5-10% can be developed to active tuberculosis; If there is not suitably treatment, an activity case can make 10-15 people newly infected every year on average, and the mortality ratio of case then surpasses 50%.If the latent infection person suffers from immunosupress simultaneously, as acquired immune deficiency syndrome (AIDS), annual just have 10% disease to send out probability.There were 8,800,000 New Development tuberculosis cases, 1,600,000 tuberculosis deaths in the whole world in 2005.Most of tuberculosis cases are in developing country, and wherein Fei Zhou the incidence of disease per capita is the highest, 28%; But case more than half is 6 Asian countries: India, China, Indonesia, Bangladesh, Pakistan, Philippine, in the Sub-Saharan Africa and some developed country, tuberculous number is on the rise, because many people's immune system is impaired because of immunosuppressant medicine, substance abuse or acquired immune deficiency syndrome (AIDS).
Spreading and ignoring the tuberculosis Control work of acquired immune deficiency syndrome (AIDS) makes tuberculosis become a kind of main infectious disease once more.In addition, multidrug resistance tuberculosis and extensive resistance to the action of a drug tuberculosis spread.The World Health Organization (WHO) announces that in 1993 tuberculosis is a healthy emergency in the whole world, and therefore diagnoses and treatment lungy becomes an instant thing and paid attention to by national governments.
Present diagnosis lungy can also can be cultivated and MTB PCR with reference to tuberculin test, tissue pathological slice, acid resistance dyeing, tulase according to clinical manifestation and X-ray.The serology aspect, enzyme linked immune assay (ELISA), chemiluminescence (CLIA), immunochromatographic method (collaurum or latex particle method), these methods all have characteristics separately and use object.Clinical manifestation and X-ray check and tuberculin experiment, tissue pathological slice or the like methods and results can be used as the goldstandard of diagnosis of tuberculosis accurately and reliably, but only is suitable for tuberculosis patient, is not suitable for just sending out to infect not seeing the symptom.
Serology detects then can early detection symptomless infection person, but ELISA test operation program complexity, false positive or false negative result appear easily, and sensitivity is low, CLIA and ELISA method are similar, just sensitivity has improved some, the problem that the not basic ELISA of solution exists, and all there is complex operation in the two, the problem that reaction time is long, and all need microplate reader or light-emitting appearance and wash complex apparatus such as plate machine and incubator, and can not single part detect, further limited their application in some basic hospitals, clinic.Having occurred both at home and abroad in recent years with collaurum or latex particle is the quick detection test paper bar of representative, but because the result is the naked eyes visualizations, is subjected to the influence of observer's subjective judgement easily, and sensitivity is low, and result precision is not high.
Magnetic immuno-chromatographic (Mgnetic ImmunoChromatographic Test, MICT) be occur in recent years the single part fast quantification detection technique of a kind of a new generation.Be to replace traditional label (collaurum, latex particle etc.) to carry out immunochromatography, be combined in biochemical substances on the super-paramagnetism nano particulate by detection detection by quantitative data to biological sample are provided with supperparamagnetic particles (superPMPs).Come the amount of presentation markup by detecting the measurement magnetic field intensity, adopt the typical curve of the immune complex-magnetic field intensity of mark, thereby reach quantitative purpose in sample area.This technology is compared with conventional art has following advantages: 1) sensitivity for analysis: than the sensitive 10-100 of all kinds of range estimation quick diagnosis methods doubly; 2) analysis speed: can in 15 seconds, measure the nearly data of 6 site of analysis; 3) linear range: in the concentration range of 1-104, be linear; 4) the used magnetic detecting instrument adopts the solid phase element, and miniaturized design is had a style of one's own, independent operating, and volume is little, and is easy and simple to handle; 5) the super-paramagnetism nano particulate can not decayed in time by polymer coating; Independently quick diagnosis chromatogram card (MAR Cassette) can directly insert the MAR detector, for the integration of many quick reagents for clinical diagnosis at present provides development space widely; But also personnel or the contingent cross pollution of instrument in the operating process, the security that has improved analysis and process have been avoided.This technology has been inherited traditional immunochromatographic method (collaurum, latex particle etc.) easy to be quick, the advantage of single part of operation, it is low to have remedied traditional immunochromatography technique sensitivity again, can only be qualitative, shortcoming that can not be quantitative, represented current real-time test (Point of Care Test, POCT) direction of technical development and trend are once appearance, development has become the first-selection that substitutes traditional immunochromatography technique at present rapidly.
Mark magnetic particle commonly used at present is a super paramagnetic particle (superPMPs), do not have any magnetic in the absence of externally-applied magnetic field, only just can show magnetic adding under the action of a magnetic field, the super paramagnetic particle of commercialization all passes through finishing, greatly facilitate labeling process, mark is easy, good reproducibility.
In recent years, because the progress of molecule clone technology, adopt multiple different expressive host to make the recombinant expressed variation of TB antigen, it is good a collection of sensitivity to have occurred, the specificity height, activity keeps HCV antigen preferably in bag quilt and the labeling process, make the reaction pattern that adopts double antigens sandwich detect TB antibody and become possibility, double antigens sandwich has highly sensitive than the detecting pattern of indirect method, specificity is good, operates easyly relatively, and the result is advantage accurately and reliably, be widely adopted human immunodeficiency virus (HIV) and microspironema pallidum (TP), just at the early-stage on the TB detection of antibodies.
Summary of the invention
Purpose of the present invention promptly is that the magnetic immuno-chromatographic technology is applied in the TB immunoassay, and the reaction pattern of creationary employing double antigens sandwich detects TB antibody.With TB antigen and rabbit igg covalent coupling on super paramagnetic particle as detecting moving phase, TB antigen and goat anti-rabbit igg bag are made detection line and nature controlling line as catching solid phase on nitrocellulose filter, carry out the detection of sample according to routine immunization chromatography ratio juris, detect in conjunction with simple and easy to do magnetism detector, realized high-sensitivity detection, combine the advantage of aforementioned several method: can single part detect, also can batch detection, and can provide quantifiable result immediately, surveying instrument is simple and reliable, easy and simple to handle, convenient and practical.
For reaching above-mentioned purpose, technical scheme of the present invention is as follows: the magnetic immuno-chromatographic test paper strip of detection TB antibody of the present invention is with coated film, the magnetic mat of particles that combines TB antigen, sample pad, adsorptive pads, sticks on the base plate successively interlaced 2mm, cover the transparent plastic diaphragm seal then on the upper strata and make, be coated with TB detection of antigens line on the wherein said coated film in advance, and the nature controlling line of goat anti-rabbit igg.Adopt the reaction pattern of double antigens sandwich to detect TB antibody.
The base plate of selecting for use is the transparent plastic base plate, and coated film is the nitrocellulose filter of 35mm width, and the adsorptive pads of selecting for use is a cellulose membrane, and the magnetic mat of particles is a fiberglass packing, and sample pad is the pretreated cellulose membrane of process sample pad treating fluid.Described sample pad treating fluid is the polyvinyl alcohol (PVA) (PVP) that contains 1%-5% casein (casein) and 0.1%-1%, and the 0.02M of 0.01-0.2% Tween-20 (Tween-20), the phosphate buffered solution of pH7.0-7.6 (PBS).
Detect the preparation method of the magnetic immuno-chromatographic test paper strip of TB antibody, may further comprise the steps:
The processing of A, antigen: select for use commercialization TB recombinant antigen (CTK company's T B fused antigen 16-14-38, cat:A6438); To 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6;
The preparation of B, magnetic particle: selecting diameter for use is the super paramagnetic particle of 100-300nm, with TB antigen and rabbit igg to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml; Selecting diameter for use is the super paramagnetic particle of 100-300nm, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, it is 20mmol/L that the adding carbodiimide makes final concentration, room temperature reaction 1 hour, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer of pH4.5-5.0 fully washs and adds TB antigen behind the magnetic particle to make the molecule ratio of TB antigen and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, use contains the 50mM of 0.1%Tween-20, and the sodium-acetate buffer washing magnetic particle of pH4.5-5.0 uses then and contains 1%PVP, 1%casein, 0.5%Tween-20, the 50mM of 5% sucrose, the boric acid of pH8.2-9.0 preserve damping fluid redissolution magnetic particle, 4 ℃ of preservations are standby, and making uses the same method is coupled to rabbit igg on the magnetic particle; The magnetic particle of TB antigen coupling and the magnetic particle of rabbit igg coupling are mixed with 2: 1 ratio, and fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed; The magnetic particle that dilution is good uses quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 10-50 μ l/cm, adds drying agent after the freeze drying and seals up for safekeeping standby.
The preparation of C, coated film: use bag to be cushioned the concentration that liquid is diluted to TB envelope antigen and goat anti-rabbit igg 0.5-2mg/m respectively, use quantitative liquid-jet device respectively with the two with the interval spray printing of 0.5-1.2cm on nitrocellulose filter, dry the back and in confining liquid, soak after 10 minutes, add drying agent and seal up for safekeeping standby in 25-35 ℃ of oven dry 8 hours;
The processing of D, sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour;
The assembling of E, test strips: interlaced successively 2mm sticks coated film, magnetic mat of particles, sample pad, adsorptive pads on the transparent plastic base plate, covers the transparent plastic diaphragm seal then on the upper strata, obtains test paper plate, and the width cutting promptly obtains test strips as requested.
Among the described step C, the preparation method of coated film is: be cushioned liquid (0.02M PB with bag, pH7.0-7.6) be 0.5mg/ml with the TB antigen diluent, the goat anti-rabbit igg dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the two with the interval spray printing of 0.8cm on nitrocellulose filter, room temperature is dried after 30 minutes and to be soaked in confining liquid after 10 minutes in 25-35 ℃ of oven dry 8 hours, adds drying agent and seals up for safekeeping standby.
Compare with existing quick detection test paper bar, the present invention has the following advantages:
Replace immunochromatography tracers such as traditional collaurum and latex particle with super paramagnetic particle, apply in the quick detection test paper bar of TB, the utilization magnetism detector carries out result's interpretation, magnetic detection value ratio according to detection line and nature controlling line carries out the yin and yang attribute judgement, reduced subjectivity, the result accurately, reliably.
The present invention is easy and simple to handle, be fit to large-scale production, detect required portable set and also go on the market, therefore can be widely used in short run or single part of unit use of using such as applying unit and some blood sampling scenes, rural area and basic unit clinic in enormous quantities such as hospital, blood station, epidemic prevention station, health check-up.Primary dcreening operation etiologic diagnosis for TB has positive meaning.
Description of drawings
Fig. 1 detects the magnetic immuno-chromatographic test paper strip structural representation of TB antibody for the present invention.
Detect magnetic immuno-chromatographic test paper strip of TB antibody and preparation method thereof for further specifying the present invention, describe especially exemplified by following embodiment, this embodiment is in order to explain rather than limit by any way the present invention.
Embodiment
The magnetic immuno-chromatographic test paper strip of TB antibody in the detection blood of the present invention, as shown in Figure 1, this test strips is at base plate 1) on interlaced successively 2mm ground paste to go up coated film 2), combine the magnetic mat of particles 3 of TB antigen), sample pad 4), adsorptive pads 5), and cover transparent plastic diaphragm seal 6 on the upper strata) test strips that assembles, coated film 2) on be coated with detection line T (TB antigen) and nature controlling line C (goat anti-rabbit igg) in advance.
In specific embodiment, the TB antigen that adopts to for commercialization antigen.The principle of utilizing double antigens sandwich to detect TB antibody detects sample, when containing TB antibody in the sample to be measured, the antigen combination of combination on antibody meeting elder generation and the magnetic particle, carrying out along with the chromatography effect, bond moves forward and arrives detection line T place, and antibody can accumulate in T line place with envelope antigen in conjunction with forming the double antigens sandwich compound once more.In addition, rabbit igg mark magnetic particle can continue to move ahead when arriving nature controlling line C, thereby goat anti-rabbit igg can combine at C line place with rabbit igg mark magnetic particle and occurs the magnetic particle aggregation equally.Entire reaction was carried out in 30 minutes fully, general reaction can be used magnetic immuno-chromatographic instrument Card Reader after 15 minutes, T line and C line all can produce corresponding magnetic signal value, calculate the ratio of T/C, get final product the yin and yang attribute of result of determination according to default boundary ratio.Whole Card Reader, calculating, with the process sequencing fully of preset bounds value comparison, magnetism detector can directly provide the yin and yang attribute result.
The preparation method of the magnetic immuno-chromatographic test paper strip of TB antibody sees following example in the detection blood of the present invention:
Embodiment 1
Detect the magnetic immuno-chromatographic test paper strip of TB antibody in the blood and the preparation method of paper box
The test strips of present embodiment and the preparation method of paper box may further comprise the steps:
The preparation of A, antigen and antibody: select commercial TB recombinant antigen for use, to 20mM, the PBS of pH7.2 (pH7.0-7.6 all is suitable for), 4 ℃ of dialysed overnight are standby.
The preparation of B, coated film:
Bag is cushioned the preparation of liquid: the phosphate buffer of 0.02M pH 7.2 (PBS) is cushioned liquid for bag, and the rearmounted 4 ℃ of preservations of 0.22 μ m filtering with microporous membrane degerming are standby, two weeks of the term of validity.
The preparation of confining liquid: contain the phosphate buffer (PBS) of the 0.02M pH7.2 (pH7.0-7.6 all is suitable for) of 0.5%BSA, it is standby that 0.22 μ m filtering with microporous membrane degerming is placed on 4 ℃ of preservations, one week of the term of validity.
The preparation of coated film: being cushioned liquid (PB of 0.02M pH7.2 (pH7.0-7.6 all is suitable for)) with bag is 0.5mg/ml with the TB antigen diluent, the goat anti-rabbit igg dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the two with the even spray printing in the interval of 0.8cm on 3.5cm width nitrocellulose filter, room temperature is dried after 30 minutes in the confining liquid (PBS that contains the 0.02M pH7.2 (pH7.0-7.6 all is suitable for) of 0.5%BSA,) in 25-35 ℃ of oven dry 8 hours, the adding drying agent was sealed up for safekeeping standby after 10 minutes in middle immersion.
The preparation of C, magnetic mat of particles:
The preparation of sodium-acetate buffer: with distilled water and sodium acetate and glacial acetic acid secure ph is 4.7 (pH4.5-5.0 all is suitable for), concentration is the acetate buffer solution of 50mM, adding Tween-20 is that 4 ℃ of preservations are standby after 0.1%, the 0.22 μ m filtering with microporous membrane degerming to final concentration, two weeks of the term of validity.
Boric acid is preserved the preparation of damping fluid: use distilled water, boric acid and borax preparation pH are 8.5 (pH8.2-9.0 all is suitable for), and final concentration is the borate buffer of 50mM, add PVP, Casine, Tween-20, sucrose, final concentration is respectively 1%, 1%, 0.5%, 5%, 0.22 4 ℃ of preservations are standby after the degerming of μ m filtering with microporous membrane, one week of the term of validity.
Selecting diameter for use is the super paramagnetic particle of 100-300nm, with TB antigen and rabbit igg to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml; Selecting diameter for use is the super paramagnetic particle of 100-300nm, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, it is 20mmol/L that the adding carbodiimide makes final concentration, room temperature reaction 1 hour, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer of pH4.5-5.0 fully washs and adds TB antigen behind the magnetic particle to make the molecule ratio of TB antigen and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, use contains the 50mM of 0.1%Tween-20, and the sodium-acetate buffer washing magnetic particle of pH4.5-5.0 uses then and contains 1%PVP, 1%casein, 0.5%Tween-20, the 50mM of 5% sucrose, the boric acid of pH8.2-9.0 preserve damping fluid redissolution magnetic particle, 4 ℃ of preservations are standby, and making uses the same method is coupled to rabbit igg on the magnetic particle; The magnetic particle of TB antigen coupling and the magnetic particle of rabbit igg coupling are mixed with 2: 1 ratio, and fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed; The magnetic particle that dilution is good uses the nozzle specially used amount with 25 μ l/cm of ioDot spray film instrument evenly to be sprayed on the glass fibre, adds drying agent after the freeze drying and seals up for safekeeping standby.
The processing of D, sample pad
1.8cm width sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour.
The sample pad treating fluid is the PBS solution that contains the PVA of 1%-5%Casein and 0.1%-1% and the 0.02M pH7.2 of 0.01-0.2%Tween-20 (pH7.0-7.6 all is suitable for).
The assembling of E, test strips and cutting
Following all operations all must carry out in temperature 20-25 ℃ the room in humidity less than 20%.
The assembling of test paper plate: use as requested that 3.5cm is the wide coated film of BioDot LM5000 type assembling instrument, 2.5cm wide thieving paper, the magnetic mat of particles that 0.8cm is wide, the wide sample pad of 1.8cm are assembled on the 9.8cm width transparent plastic base plate, stick upper strata transparent plastic cover plate, be assembled into test paper plate.
Cutting of test strips: use BioDot CM4000 type cutting cutter that the test paper plate that assembles is cut into the wide finished product test strips of 0.5cm.
The assembling of F, test card
The single part test strips of well cutting of the present invention is placed in the draw-in groove on the plastic bottom card, covers loam cake, use card press machine up and down two plastic clips compress, guarantee that whole test strips is in tensioned state.Adding the drying agent room temperature seals up for safekeeping standby.
G, determine the 2 D code information of this batch
The name of an article: TB antibody magnetic test card
Batch: on the assembling date of test card, form is: Year/Month/Day, XXXX/XX/XX
Determining of yin and yang attribute interpretation standard: get 100 parts and confirm TB antibody positive sample (power all has), 500 parts at random sample use this batch test card to detect, use the magnetism detector testing result, calculate the T/C value of each test card, use statistical method computation of mean values and standard deviation, determine: T/C＜0.1 is negative.T/C＞0.2 is positive, is gray area between the two.
The printing of H, two-dimension code is pasted
With in the above-mentioned 2 D code information input two-dimension code printer and print, two-dimension code is pasted on the ad-hoc location of test card, use two-dimension code paste position detecting device to inspect 2% at random by random samples and guarantee that two-dimension code pastes errorless.
I, finished product packing
The single part test card and that the posts two-dimension code drying prescription of being responsible for a task until it is completed is sealed in the aluminium foil bag, 100 person-portions are that a packing places in the packing box, and a instructions of a box and 1 bottle of 10ml dress chromatography damping fluid are promptly made paper box, this paper box keeps in Dark Place in room temperature, and the shelf-life is 18 months.The chromatography buffer formulation is: 1%Tween-20, and 0.5%Triton X-100,1%NP-40,0.05%NaN3, the PBS of 20mmol pH7.2 (pH7.0-7.6 all is suitable for).
In the step except the preparation of magnetic particle: the molecule ratio that makes TB antigen and magnetic particle is 2: 1.Other step is with embodiment 1,
In the step except the preparation of magnetic particle: the molecule ratio that makes TB antigen and magnetic particle is 10: 1.Other step is with embodiment 1.
Except behind TB antigen and the rabbit igg mark magnetic particle blend step in: fully use behind the mixing and preserve damping fluid and with 1: 10 ratio mixture diluted fiberglass packing to be sprayed is used, other step is with embodiment 1.
Except behind TB antigen and the rabbit igg mark magnetic particle blend step in: fully use behind the mixing and preserve damping fluid and with 1: 20 ratio mixture diluted fiberglass packing to be sprayed is used, other step is with embodiment 1.
The using method of test card of the present invention
1, application of sample
From packing box, take out single part test card, tear the aluminium foil strip packing, test card is placed on the smooth desktop, get 50 μ l sample serum with micropipettor and add in the well on the card, add 50 μ l chromatography damping fluids again, wait question response to carry out 15 minutes.
2, measurement and result output
The MICT detector is started shooting in advance, test card is inserted the card inserting mouth of detector, the operation instrument, instrument can read the 2 D code information on the card automatically and measure, and prints measurement result immediately, and the yin and yang attribute result can show in print result.
Claims (3)
1. magnetic immuno-chromatographic test paper strip that detects TB antibody, it is characterized in that: this test strips is that coated film, the magnetic mat of particles that combines TB antigen, sample pad, adsorptive pads are sticked on the base plate successively interlaced 2mm, cover the transparent plastic diaphragm seal then on the upper strata and assemble, be coated with TB Detection of antigen line and nature controlling line on the wherein said coated film in advance.
2. the magnetic immuno-chromatographic test paper strip of detection TB antibody according to claim 1 is characterized in that: adopt the reaction pattern of double antigens sandwich to detect TB antibody.
3. the preparation method of the magnetic immuno-chromatographic test paper strip of detection TB antibody according to claim 1 is characterized in that: may further comprise the steps:
1) processing of antigen: select the commercialization TB reorganization pairing antigen of technique for gene engineering preparation for use, to 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6;
2) preparation of coated film: use 0.02mM, the PBS of pH7.0-7.6, respectively TB envelope antigen and goat anti-rabbit igg are diluted to the concentration of 0.5-1.5mg/ml, use quantitative liquid-jet device respectively with the two with the interval spray printing of 0.5-1.2cm on nitrocellulose filter, dry the back and in confining liquid, soak after 10 minutes, add drying agent and seal up for safekeeping standby in 25-35 ℃ of oven dry 8 hours;
3) preparation of magnetic mat of particles: with TB antigen and rabbit igg to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml; Selecting diameter for use is the super paramagnetic particle of 100-300nm, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, it is 20mmol/L that the adding carbodiimide makes final concentration, room temperature reaction 1 hour, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer of pH4.5-5.0 fully washs and adds TB antigen behind the magnetic particle to make the molecule ratio of TB antigen and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, use contains the 50mM of 0.1%Tween-20, and the sodium-acetate buffer washing magnetic particle of pH4.5-5.0 uses then and contains 1%PVP, 1%casein, 0.5%Tween-20, the 50mM of 5% sucrose, the boric acid of pH8.2-9.0 preserve damping fluid redissolution magnetic particle, 4 ℃ of preservations are standby, and making uses the same method is coupled to rabbit igg on the magnetic particle; The magnetic particle of TB antigen coupling and the magnetic particle of rabbit igg coupling are mixed with 2: 1 ratio, and fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed; The magnetic particle that dilution is good uses quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 10-50 μ l/cm, adds drying agent after the freeze drying and seals up for safekeeping standby;
4) processing of sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour; Described sample pad treating fluid is the polyvinyl alcohol (PVA) that contains 0.5%-2.5%BSA and 0.1%-1%, and the 0.02M of 0.01-0.2% Tween-20, the phosphate buffered solution of pH7.0-7.6;
5) assembling of test strips: interlaced successively 2mm sticks coated film, magnetic mat of particles, sample pad, adsorptive pads on the transparent plastic base plate, covers the transparent plastic diaphragm seal then on the upper strata and obtains test paper plate, and width cutting as requested promptly obtains test strips.
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