CN105137072B - A kind of mycobacterium tuberculosis LAM detection kit, preparation method and using method - Google Patents
A kind of mycobacterium tuberculosis LAM detection kit, preparation method and using method Download PDFInfo
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- CN105137072B CN105137072B CN201510218677.7A CN201510218677A CN105137072B CN 105137072 B CN105137072 B CN 105137072B CN 201510218677 A CN201510218677 A CN 201510218677A CN 105137072 B CN105137072 B CN 105137072B
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
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Abstract
The present invention relates to a kind of Mycobacterium tuberculosis LAM (LMA) detection kit based on nano immune magnetic bead near-infrared fluorescent labelling method.In this test kit, target the monoclonal antibody of LAM near infrared fluorescent dye labelling, the polyclonal antibody targeting LAM is coated on nanometer magnetic bead surface.Using double antibody sandwich method principle, Magnetic Isolation conjugate and educt, detect the fluorescence intensity of magnetic conjugate using portable high sensitivity low noise excitation formula fluorescence detector, thus detecting LAM content in measuring samples.LAM detection be applied to clinical pathology specimen for the present invention, has the characteristics that quick, sensitive, anti-spuious fluorescence interference, transmitting fluorescence holding time are long.
Description
Technical field
The invention belongs to biological field is and in particular to a kind of Mycobacterium tuberculosis LAM (LMA) detects
Test kit, preparation method and using method.
Technical background
The present invention establishes a kind of nanometer magnetic bead-near-infrared fluorescent of detection mycobacterium tuberculosis thalline Related Component LAM
Labelling-detection kit.Including the near infrared fluorescent dye labeling method of monoclonal antibody and the polyclonal antibody of nanometer magnetic bead
Method for coating and serial related reagent.The present invention, using near infrared fluorescent dye as labelling, using immunoassay principle, is prepared
Detection LAM double antibody sandwich method Near-infrared Fluorescence Probe Reagents box.Magnetic bead-combination is carried out using Portable mobile sample introduction fluorescence detector
Thing fluorescent strength determining.During detection by quantitative, pattern detection value is substituted into normal equation, you can analysis detection sample target protein
Concentration；Qualitative detection then using 2 times of the fluorescence intensity of the negative control cutoff values as reaction system, more than 2 times of cutoff
It is worth for the positive, otherwise be then feminine gender.The detection of this test kit mycobacterium tuberculosis thalline LAM be applied to pathology sample.This
Bright have the characteristics that quick, Gao Min, take into account qualitative and accurate quantitative.
Tuberculosis are a kind of ancient infectious diseases, and the current whole world has 1/3 population to be felt by mycobacterium tuberculosis
Dye, has nearly 8,000,000 people to die from pulmonary tuberculosis every year；Drug resistance and a large amount of appearance of multi-drug resistant bacterial strain, are control lungy again
System brings bigger difficulty；More researchs also demonstrate that mycobacterium tuberculosis can strengthen the duplication of HIV-1 virus, therefore in AIDS
In sick the infected, double infection person is numerous, becomes and leads to AIDS patient main causes of death.The current TB patient populations of China
Occupy second place of the world, be that 22 TB height bear one of countries, China's the 4th TB Survey on epidemiological features money in 2000 in the world
Material display, China's MTB infection rate is 44.5%, activeness TB patient 4,510,000, and annual death toll reaches 130,000.
Measuring means for mycobacterium tuberculosis mainly have smear for microscopic examination method at present, and the method needs testing staff to have
Skilled inspection technical ability, method detection sensitivity is low, typically needs to exist 10 in specimen3CFU/ml antibacterial just has 10～15%
Positive rate, and antibacterial be cannot be carried out accurately identify；Culture method, mainly carries out this work, detection in infectious hospital
Cycle is longer, generally requires 6 time-of-weeks and just can transmit messages announcement, although the full automatic microorganism assessing instrument in recent years occurring substantially reduces
Round of visits, it usually needs 2 weeks about time, this does not solve the problems, such as efficiency yet at all, and there is with high costs lacking
Fall into, simple reagent cost is in 120 yuan/test, and needs large-scale equipment；Serologic test method is concentrated mainly on to tuberculosis
The detection of mycobacteria associated antibodies, because tuberculosis infection patient has hypoimmunity, does not often express related anti-
Body, or because adult's infection tulase probability is many, due to immunological memory, once the infected also can detect that associated antibodies to many,
This just causes trouble for judgement；Recently wide model applies to Clinical detection, such as PCR method, probe in detecting to the means of molecular biology
Method etc., these method specificitys are good, but need special detecting instrument, promote the use existing problems, the sensitivity of superelevation also carries
Carry out the problem-false positive being difficult to overcome；Tuberculosis infection T cell spot test is used for detecting specific T-cells after tuberculosis infection
The gamma interferon of secretion, and result judges whether to have infected tuberculosis accordingly, the method feature is loaded down with trivial details, and high cost should not push away
Extensively, mainly make examination in western countries at present to use；The amplification test of mycobacterium tuberculosis specific bacteriophage is also once in tuberculosis
Laboratory played effect, there is also loaded down with trivial details, high cost, the shortcoming needing enough experiences in result judges.The method
False positive rate is 3%～7%, and false negative rate is 15%～40%.Positive coincidence rate with culture is 75%～95%, specificity
For 88%～99%.
Nanometer magnetic bead method is combined by the present invention with near-infrared fluorescent labelling method, using the activation of nanometer magnetic bead surface carboxyl groups
Can be combined with the amino covalence of antibody surface afterwards, thus antibody is fixed to nanometer magnetic bead surface, play nanometer magnetic bead and compared table
Area is big, and adsorptivity is strong, and suspension stability is good, is conducive to the feature that antigen antibody reaction is smoothed out；Near-infrared fluorescent simultaneously
Dyestuff has the advantages that sensitivity height, selectivity are good, and its exciting light and emissioning light spectrum, can be maximum all near infrared region
The advantages of limit reduces the interference of spuious fluorescent material in environment.Set up the near-infrared fluorescent of mycobacterium tuberculosis thalline LAM
Labelling-nano magnetic bead detection method, LAM detection limitation reaches 1.0ng/ml level.
Content of the invention
In order to overcome drawbacks described above, provide technical scheme below：
A kind of mycobacterium tuberculosis LAM detection kit based on nano immune magnetic bead-near-infrared fluorescent labelling method, described
Test kit is by the monoclonal antibody of near infrared fluorescent dye labelling, the coated nanometer magnetic bead of polyclonal antibody, magnetic separator, egg
White standard substance, buffer composition；Wherein, the monoclonal antibody of near infrared fluorescent dye labelling is the anti-LAM of rabbit；It is coated nanometer magnetic bead
Polyclonal antibody be rabbit multi-resistance LAM.The work set up between L-arabinose standard substance gradient concentration and Fluorescence emission values is bent
Line, measured transmitting fluorescence intensity substitutes into (qualitative) detection of quantitation that equation can complete desired polysaccharide content in sample.
Preferably, described near infrared fluorescent dye is a length of 777nm of excitation light wave, and wavelength of transmitted light is the fluorescence of 790nm
The near infrared fluorescent dye DyLight800 of molecule, preferably NHS activation.
A kind of described mycobacterium tuberculosis LAM detectable based on nano immune magnetic bead-near-infrared fluorescent labelling method
The preparation method of box, step is as follows：
(1) prepare the LAM monoclonal antibody of near infrared fluorescent dye labelling；
Dylight800 (purchased from Thermofisher company) is used PBS (PH7.4) to dilute 10 times, takes 1.4 μ L
After LAM monoclonal antibody (1mg/ml) (purchased from Mossman Associates Inc company) anti-with 20 μ g rabbits softly mixs homogeneously
React 2 hours in room temperature lucifuge；After reaction terminates, marked product is put in bag filter, 4 DEG C, PBS is dialysed 4 hours.
Add final concentration of 1.5%BSA and 0.1%Tween20, Hydrazoic acid,sodium salt 0.1 ‰, 4 DEG C of preservations in the good antibody-solutions of labelling；Make
With front, marked product is diluted 2000 times with PBS；
(2) preparation of LAM polyclonal antibody
1) purification of LAM：
MTB H37RV is seeded on Roche egg medium, cultivates 3～4 weeks for 37 DEG C, collect bacterium colony and be placed in 5ml EP pipe
In, 100 DEG C of water-baths inactivate 2 hours, and with 2mlPBS (PH7.4) buffer solution 2 times, supernatant (8000r/min) is abandoned in centrifugation；Add
2ml chloroform/methanol (2: 1) mixed liquor defat 3 times, supernatant (8000r/min) is abandoned in centrifugation；2mlPBS (PH7.4) is added to suspend, ice
Bath ultrasonication (ultrasonic 10s, stops 10s, common 30min), 8000r/min centrifuging and taking supernatant, add the phenol extraction 70 of 2ml40%
DEG C, 1 hour, 8000r/min centrifuging and taking supernatant, dialysed 2 days with distilled water, using Sephacryl S-100 gel filtration chromatography, use
NaCl (0.05mmol/L) eluant solution, flow velocity 0.5ml/min.EP pipe is collected.
2) quantitation of LAM：
Take concentrated sulphuric acid 1ml, 9% phenol 200 μ l, specimen 200 μ l, after mixing, lucifuge is reacted 30 minutes, blank zeroing, with L-
Arabinose is many Standard for Sugars, makes the standard curve of concentration range 0.1～3.0mg/ml, surveys absorbance (A485) value, straight line
Polyoses content in regression Calculation specimen；
3) LAM polyclonal antibody preparation：
With the LAM of this laboratory preparation as immunogen, entrust Southern Yangtze University's Foodstuffs Academy on behalf of preparation LAM Anti-TNF-α
Body, the antibody after making is rabbit anti-LAM polyclonal antibody, and concentration is 1mg/ml.
(3) the preparation coated nanometer magnetic bead of LAM polyclonal antibody is (purchased from Wuhan Wawasye Technology Development Co., Ltd. north
Capital biotechnology research institute)；
1) pretreatment of nanometer magnetic bead：
Gently mix bead suspension, draw 0.01ml suspension (25mg/ml) and add in 1.5ml EP test tube；Add
0.1ml redistilled water, gently mixes and mounts test tube on magnetic sheet, standing Aspirate supernatant after 2 minutes；Repeat the above steps 1 time；
Add the ethyl sulfonic acid solution (pH=5.0, MES) of 1ml 0.1M, resuspended magnetic bead；
2) activation of nanometer magnetic bead
Above-mentioned EP pipe is placed on magnetic sheet, is washed with 1mlMES 2 times, abandon supernatant；
Configure the MES of final concentration of 0.1M before use, it includes the concentration of EDC and NHS and is 40g/L (addition above-mentioned two
After planting material, it is placed on agitator abundant mixing 15 minutes, stand-by).With the resuspended magnetic bead of this liquid；
Adsorb magnetic bead with magnetic sheet, abandon supernatant, then cleaned with MES once, after absorption magnetic bead, abandon supernatant；
3) the linking of magnetic bead and albumen；
Add 50 μ L LAM polyclonal antibody (1mg/ml) and 500 μ L PBS (PH5.5) in processed good magnetic bead, mix
Even, room temperature, it is placed in sustained oscillation on shaking table, be incubated 2 hours；
Cleaned 3 times with PBS (PH5.5,0.05%Tween), abandon supernatant；
Add 1ml Tris buffer (0.1M, 0.1%BSA), mix, room temperature, be placed in sustained oscillation on shaking table, be incubated 2
Hour；
Add 1ml PBS (PH5.5,0.05%Tween), stand 2 minutes, abandon supernatant；It is repeated 3 times, add 1ml
PBS (PH5.5), abandons supernatant, is repeated 3 times.When abandoning supernatant, test tube need to be placed on magnetic sheet；
Add 1ml PBS (pH=7.4,0.05%Tween-20,0.1%BSA, 0.05NaN3), stand-by；
(4) LAM serial standards are prepared；
LAM (this room prepare, 1mg/ml), PBS (PH7.4) buffer is diluted to 0.5,1.0,2.5,5.0,10.0,
20.0ng/ml.
(1) the LAM standard substance respectively taking each concentration of 50 μ L, in EP pipe, separately take 1 EP pipe to add 50 μ LPBS (PH7.4) to delay
Rush liquid as negative control；
(2) each pipe more than adds fluorescently-labeled LAM monoclonal antibody 50 μ L, and 37 DEG C are reacted 30 minutes；
(3) take into pretreatment nano immune magnetic bead 100 μ L, be diluted to 1000 μ L with PBS (PH7.4)；Draw
50 μ L immunomagnetic beadses, add in described test tube, and 37 DEG C are reacted 15 minutes；Add 500 μ LddH2O, magnetic sheet stands 2 minutes, abandons
Supernatant, repeats this step 3 time；
The processing method of described pretreatment nano immune magnetic bead is：
The preparation coated nanometer magnetic bead of LAM polyclonal antibody is (purchased from Wuhan Wawasye Technology Development Co., Ltd. Beijing
Biotechnology research institute)：
1) pretreatment of nanometer magnetic bead：
Gently mix bead suspension, draw 0.01ml suspension (25mg/ml) and add in 1.5ml EP test tube；Add
0.1ml redistilled water, gently mixes and mounts test tube on magnetic sheet, standing Aspirate supernatant after 2 minutes；Repeat the above steps 1 time；
Add the ethyl sulfonic acid solution (pH=5.0, MES) of 1ml 0.1M, resuspended magnetic bead；
2) activation of nanometer magnetic bead
Above-mentioned EP pipe is placed on magnetic sheet, is washed with 1mlMES 2 times, abandon supernatant；
Configure the MES of final concentration of 0.1M before use, it includes the concentration of EDC and NHS and is 40g/L (addition above-mentioned two
After planting material, it is placed on agitator abundant mixing 15 minutes, stand-by).With the resuspended magnetic bead of this liquid；
Adsorb magnetic bead with magnetic sheet, abandon supernatant, then cleaned with MES once, after absorption magnetic bead, abandon supernatant；
3) the linking of magnetic bead and albumen；
Add 50 μ L LAM polyclonal antibody (1mg/ml) and 500 μ L PBS (PH5.5) in processed good magnetic bead, mix
Even, room temperature, it is placed in sustained oscillation on shaking table, be incubated 2 hours；
Cleaned 3 times with PBS (PH5.5,0.05%Tween), abandon supernatant；
Add 1ml Tris buffer (0.1M, 0.1%BSA), mix, room temperature, be placed in sustained oscillation on shaking table, be incubated 2
Hour；
Add 1ml PBS (PH5.5,0.05%Tween), stand 2 minutes, abandon supernatant；It is repeated 3 times, add 1ml
PBS (PH5.5), abandons supernatant, is repeated 3 times.When abandoning supernatant, test tube need to be placed on magnetic sheet；
Add 1ml PBS (pH=7.4,0.05%Tween-20,0.1%BSA, 0.05NaN3), stand-by；
(4) each pipe adds 100 μ L PBS (PH7.4), mixes, with Portable mobile sample introduction fluorescence detector (is
Model machine prepared by Chinese Academy of Sciences's Theoretical Physics, this equipment is cured as a length of 777nm of excitation light wave, and wavelength of transmitted light is 790nm) enter
Row fluorescent strength determining；
(5) result judges
Qualitative detection is using 2 times of negative control fluorescence intensity level as Cutoff value；
Detection by quantitative carries out regression analyses with fluorescent emission intensity and corresponding LAM standard concentration, Criterion curve,
And fit equation.
Beneficial effect：
1st, the fluorescence lifetime of typical organic fluorescent dye is only several nanoseconds (ns), and this is spontaneous glimmering with a lot of biological specimens
The optical attenuation time is suitable.And the near-infrared fluorescent life-span be up to few tens of nano-seconds (20-50ns), this make light excite several nanoseconds with
Afterwards, most autofluorescence background oneself through decay, and quantum dot fluorescence yet suffers from, and now can obtain no ambient interferences
Fluorescence signal.Further, since the molar extinction coefficient of near infrared fluorescent dye exceeds 10-50 than common organic fluorescent dye
Times, the fluorescence intensity that it is launched is 10-20 times of organic dyestuff.
2nd, using nanometer magnetic bead as being coated carrier, advantage is increased the contact area of antigen-antibody, increased anti-
Answer chance, simultaneously because reaction is to carry out in liquid environment, is conducive to the space conformation of protein to launch, can greatly speed up
Response speed, reduces the response time.
3rd, adopt nanometer magnetic bead as reaction environment medium, also can for effective sharp separation offer of conjugate and educt
Energy.
Brief description
Fig. 1 LAM examination criteria curve chart
In figure, X-axis：Fluorescent emission intensity；Y-axis：Standard concentration
Specific embodiment
The present invention will combine accompanying drawing and will be described further by following examples.Embodiment：
A kind of mycobacterium tuberculosis LAM detection kit based on nano immune magnetic bead-near-infrared fluorescent labelling method, described
Test kit is by the monoclonal antibody of near infrared fluorescent dye labelling, the coated nanometer magnetic bead of polyclonal antibody, magnetic separator, egg
White standard substance, buffer composition；Wherein, the monoclonal antibody of near infrared fluorescent dye labelling is the anti-LAM of rabbit；It is coated nanometer magnetic bead
Polyclonal antibody be rabbit multi-resistance LAM.The work set up between L-arabinose standard substance gradient concentration and Fluorescence emission values is bent
Line, measured transmitting fluorescence intensity substitutes into (qualitative) detection of quantitation that equation can complete desired polysaccharide content in sample.
Preferably, described near infrared fluorescent dye is a length of 777nm of excitation light wave, and wavelength of transmitted light is the fluorescence of 790nm
The near infrared fluorescent dye DyLight800 of molecule, preferably NHS activation.
A kind of described mycobacterium tuberculosis LAM detectable based on nano immune magnetic bead-near-infrared fluorescent labelling method
The preparation method of box, step is as follows：
(1) prepare the LAM monoclonal antibody of near infrared fluorescent dye labelling；
Dylight800 (purchased from Thermofisher company) is used PBS (PH7.4) to dilute 10 times, takes 1.4 μ L
After LAM monoclonal antibody (1mg/ml) (purchased from Mossman Associates Inc company) anti-with 20 μ g rabbits softly mixs homogeneously
React 2 hours in room temperature lucifuge；After reaction terminates, marked product is put in bag filter, 4 DEG C, PBS is dialysed 4 hours.
Add final concentration of 1.5%BSA and 0.1%Tween20, Hydrazoic acid,sodium salt 0.1 ‰, 4 DEG C of preservations in the good antibody-solutions of labelling；Make
With front, marked product is diluted 2000 times with PBS；
(2) preparation of LAM polyclonal antibody
1) purification of LAM：
MTB H37RV is seeded on Roche egg medium, cultivates 3～4 weeks for 37 DEG C, collect bacterium colony and be placed in 5mlEP pipe
In, 100 DEG C of water-baths inactivate 2 hours, and with 2mlPBS (PH7.4) buffer solution 2 times, supernatant (8000r/min) is abandoned in centrifugation；Add
2ml chloroform/methanol (2: 1) mixed liquor defat 3 times, supernatant (8000r/min) is abandoned in centrifugation；2mlPBS (PH7.4) is added to suspend, ice
Bath ultrasonication (ultrasonic 10s, stops 10s, common 30min), 8000r/min centrifuging and taking supernatant, add the phenol extraction 70 of 2ml40%
DEG C, 1 hour, 8000r/min centrifuging and taking supernatant, dialysed 2 days with distilled water, using Sephacryl S-100 gel filtration chromatography, use
NaCl (0.05mmol/L) eluant solution, flow velocity 0.5ml/min.EP pipe is collected.
2) quantitation of LAM：
Take concentrated sulphuric acid 1ml, 9% phenol 200 μ l, specimen 200 μ l, after mixing, lucifuge is reacted 30 minutes, blank zeroing, with L-
Arabinose is many Standard for Sugars, makes the standard curve of concentration range 0.1～3.0mg/ml, surveys absorbance (A485) value, straight line
Polyoses content in regression Calculation specimen；
3) LAM polyclonal antibody preparation：
With the LAM of this laboratory preparation as immunogen, entrust Southern Yangtze University's Foodstuffs Academy on behalf of preparation LAM Anti-TNF-α
Body, the antibody after making is rabbit anti-LAM polyclonal antibody, and concentration is 1mg/ml.
(3) the preparation coated nanometer magnetic bead of LAM polyclonal antibody is (purchased from Wuhan Wawasye Technology Development Co., Ltd. north
Capital biotechnology research institute)；
1) pretreatment of nanometer magnetic bead：
Gently mix bead suspension, draw 0.01ml suspension (25mg/ml) and add in 1.5ml EP test tube；Add
0.1ml redistilled water, gently mixes and mounts test tube on magnetic sheet, standing Aspirate supernatant after 2 minutes；Repeat the above steps 1 time；
Add the ethyl sulfonic acid solution (pH=5.0, MES) of 1ml 0.1M, resuspended magnetic bead；
2) activation of nanometer magnetic bead
Above-mentioned EP pipe is placed on magnetic sheet, is washed with 1mlMES 2 times, abandon supernatant；
Configure the MES of final concentration of 0.1M before use, it includes the concentration of EDC and NHS and is 40g/L (addition above-mentioned two
After planting material, it is placed on agitator abundant mixing 15 minutes, stand-by).With the resuspended magnetic bead of this liquid；
Adsorb magnetic bead with magnetic sheet, abandon supernatant, then cleaned with MES once, after absorption magnetic bead, abandon supernatant；
3) the linking of magnetic bead and albumen；
Add 50 μ L LAM polyclonal antibody (1mg/ml) and 500 μ L PBS (PH5.5) in processed good magnetic bead, mix
Even, room temperature, it is placed in sustained oscillation on shaking table, be incubated 2 hours；
Cleaned 3 times with PBS (PH5.5,0.05%Tween), abandon supernatant；
Add 1ml Tris buffer (0.1M, 0.1%BSA), mix, room temperature, be placed in sustained oscillation on shaking table, be incubated 2
Hour；
Add 1ml PBS (PH5.5,0.05%Tween), stand 2 minutes, abandon supernatant；It is repeated 3 times, add 1ml
PBS (PH5.5), abandons supernatant, is repeated 3 times.When abandoning supernatant, test tube need to be placed on magnetic sheet；
Add 1ml PBS (pH=7.4,0.05%Tween-20,0.1%BSA, 0.05NaN3), stand-by；
(4) LAM serial standards are prepared；
LAM (this room prepare, 1mg/ml), PBS (PH7.4) buffer is diluted to 1,5,25,125,625ng/ml.
(1) the LAM standard substance respectively taking each concentration of 50 μ L, in EP pipe, separately take 1 EP pipe to add 50 μ LPBS (PH7.4) to delay
Rush liquid as negative control；
(2) each pipe more than adds fluorescently-labeled LAM monoclonal antibody 50 μ L, and 37 DEG C are reacted 30 minutes；
(3) take into pretreatment nano immune magnetic bead 100 μ L, be diluted to 1000 μ L with PBS (PH7.4)；Draw
50 μ L immunomagnetic beadses, add in described test tube, and 37 DEG C are reacted 15 minutes；Add 500 μ LddH2O, magnetic sheet stands 2 minutes, abandons
Supernatant, repeats this step 3 time；
The processing method of described pretreatment nano immune magnetic bead is：
The preparation coated nanometer magnetic bead of LAM polyclonal antibody is (purchased from Wuhan Wawasye Technology Development Co., Ltd. Beijing
Biotechnology research institute)：
1) pretreatment of nanometer magnetic bead：
Gently mix bead suspension, draw 0.01ml suspension (25mg/ml) and add in 1.5ml EP test tube；Add
0.1ml redistilled water, gently mixes and mounts test tube on magnetic sheet, standing Aspirate supernatant after 2 minutes；Repeat the above steps 1 time；
Add the ethyl sulfonic acid solution (pH=5.0, MES) of 1ml 0.1M, resuspended magnetic bead；
2) activation of nanometer magnetic bead
Above-mentioned EP pipe is placed on magnetic sheet, is washed with 1mlMES 2 times, abandon supernatant；
Configure the MES of final concentration of 0.1M before use, it includes the concentration of EDC and NHS and is 40g/L (addition above-mentioned two
After planting material, it is placed on agitator abundant mixing 15 minutes, stand-by).With the resuspended magnetic bead of this liquid；
Adsorb magnetic bead with magnetic sheet, abandon supernatant, then cleaned with MES once, after absorption magnetic bead, abandon supernatant；
3) the linking of magnetic bead and albumen；
Add 50 μ L LAM polyclonal antibody (1mg/ml) and 500 μ L PBS (PH5.5) in processed good magnetic bead, mix
Even, room temperature, it is placed in sustained oscillation on shaking table, be incubated 2 hours；
Cleaned 3 times with PBS (PH5.5,0.05%Tween), abandon supernatant；
Add 1ml Tris buffer (0.1M, 0.1%BSA), mix, room temperature, be placed in sustained oscillation on shaking table, be incubated 2
Hour；
Add 1ml PBS (PH5.5,0.05%Tween), stand 2 minutes, abandon supernatant；It is repeated 3 times, add 1ml
PBS (PH5.5), abandons supernatant, is repeated 3 times.When abandoning supernatant, test tube need to be placed on magnetic sheet；
Add 1ml PBS (pH=7.4,0.05%Tween-20,0.1%BSA, 0.05NaN3), stand-by；
(4) each pipe adds 100 μ L PBS (PH7.4), mixes, with Portable mobile sample introduction fluorescence detector (is
Model machine prepared by Chinese Academy of Sciences's Theoretical Physics, this equipment is cured as a length of 777nm of excitation light wave, and wavelength of transmitted light is 790nm) enter
Row fluorescent strength determining；
(5) result judges
Qualitative detection is using 2 times of negative control fluorescence intensity level as Cutoff value；
Detection by quantitative carries out regression analyses with fluorescent emission intensity and corresponding LAM standard concentration, Criterion curve,
And fit equation.
Instantiation is：LAM in detection hydrothorax
5ml hydrothorax is taken to carry out supersound process, ultrasonic degradation mode of operation is：Work 5s, rest 10s, Φ 3mm horn,
60% energy, 30 circulations altogether.
Standard substance are diluted to each concentration specified in description, after taking 50 μ L standard substance, blank and process respectively
Hydrothorax, adds A liquid 50 μ L, and 37 DEG C are reacted 30 minutes；
Add B liquid 50 μ L, 37 DEG C are reacted 15 minutes.Add 500 μ L redistilled waters, magnetic frame stand 2 minutes, abandons supernatant,
Repeat this step 3 time.
Each pipe adds 100 μ L PBS (PH7.4), carries out fluorescence intensity with Portable mobile sample introduction fluorescence detector
Measure.
Result judges
Qualitative detection is using 2 times of negative control fluorescence intensity level as Cutoff value；
Detection by quantitative carries out regression analyses with fluorescent emission intensity and corresponding standard concentration, Criterion curve, and
Fit equation.
Table 1：Variable concentrations LAM standard substance correspond to testing result
It is indicated above that the parameters in the inventive method are all optimum selections, the optimal effectiveness of the achievable present invention.
The above, be only presently preferred embodiments of the present invention, is not the restriction that the present invention is made with other forms, appoints
What those skilled in the art possibly also with the disclosure above technology contents changed or be modified as equivalent variations etc.
Effect embodiment.But every without departing from technical solution of the present invention content, according to the present invention technical spirit to above example institute
Any simple modification, equivalent variations and the remodeling made, still falls within the protection domain of technical solution of the present invention.
Claims (1)
1. a kind of mycobacterium tuberculosis LAM detection kit based on nano immune magnetic bead-near-infrared fluorescent labelling method, its feature
It is：Described test kit is by the monoclonal antibody of near infrared fluorescent dye labelling, the coated nanometer magnetic bead of polyclonal antibody, magnetic
Separator, protein standard substance, buffer composition；Wherein, the monoclonal antibody of near infrared fluorescent dye labelling is the anti-LAM of rabbit；Bag
It is rabbit multi-resistance LAM by the polyclonal antibody of nanometer magnetic bead；Set up the work between LAM standard substance gradient concentration and Fluorescence emission values
Curve, measured transmitting fluorescence intensity substitutes into the detection by quantitative that equation can complete desired polysaccharide content in sample；
The preparation side of the described mycobacterium tuberculosis LAM detection kit based on nano immune magnetic bead-near-infrared fluorescent labelling method
Method, step is as follows：
(1) prepare the LAM monoclonal antibody of near infrared fluorescent dye labelling；
The PBS that Dylight800 pH value purchased from Thermofisher company is 7.4 dilutes 10 times, takes 1.4 μ L
It is that 1mg/ml, the anti-LAM monoclonal antibody of 20 μ g rabbits softly mix with the concentration purchased from Mossman Associates Inc company
Uniformly react 2 hours after room temperature lucifuge；After reaction terminates, marked product is put in bag filter, 4 DEG C, PBS is dialysed
4 hours；Add final concentration of 1.5%BSA and 0.1%Tween20 in the good antibody-solutions of labelling, Hydrazoic acid,sodium salt 0.1 ‰, 4 DEG C
Preserve；Using front, marked product is diluted 2000 times with PBS；
(2) preparation of LAM polyclonal antibody
1) purification of LAM：
MTB H37RV is seeded on Roche egg medium, cultivates 3～4 weeks for 37 DEG C, collect bacterium colony and be placed in 5mlEP pipe,
100 DEG C of water-baths inactivate 2 hours, are washed 2 times with the PBS that the pH value of 2ml is 7.4, and 8000r/min, 10min centrifugation is abandoned
Clearly；Add 2ml chloroform: methanol=2: 1 mixed liquor defat 3 times, supernatant is abandoned in 8000r/min, 10min centrifugation；Add the pH value of 2ml
PBS for 7.4 suspends, and ice-bath ultrasonic crushes：Ultrasonic 10s, stops 10s, common 30min, 8000r/min, 10min, centrifuging and taking supernatant,
The phenol adding 2ml40% extracts, 70 DEG C, 1 hour, and 8000r/min, 10min centrifuging and taking supernatant is dialysed 2 days with distilled water, adopted
With SepHacryl S-100 gel filtration chromatography, with the NaCl solution eluting of 0.05mmol/L, flow velocity 0.5ml/min, received with EP pipe
Collection 2ml/ pipe；
2) quantitation of LAM：
Take concentrated sulphuric acid 1ml, 9% phenol 200 μ l, specimen 200 μ l, after mixing, lucifuge is reacted 30 minutes, blank zeroing, with L- I
Uncle's sugar is many Standard for Sugars, makes the standard curve of concentration range 0.1～3.0mg/ml, surveys absorbance A485Value, rectilinear regression calculates
Polyoses content in specimen；
3) LAM polyclonal antibody preparation：
With the LAM of this laboratory preparation as immunogen, entrust Southern Yangtze University's Foodstuffs Academy on behalf of preparation LAM polyclonal antibody, system
Antibody after one-tenth is rabbit anti-LAM polyclonal antibody, and concentration is 1mg/ml；
(3) prepare the coated nanometer magnetic bead of LAM polyclonal antibody, purchased from Wuhan Wawasye Technology Development Co., Ltd.'s Beijing life
Thing technical research institute；
1) pretreatment of nanometer magnetic bead：
Gently mix bead suspension, the 25mg/ml suspension drawing 0.01ml adds in 1.5ml EP test tube；Add
0.1ml redistilled water, gently mixes and mounts test tube on magnetic sheet, standing Aspirate supernatant after 2 minutes；Repeat the above steps 1 time；
Add the ethyl sulfonic acid solution that 1ml 0.1M, pH are 5.0, resuspended magnetic bead；
2) activation of nanometer magnetic bead
Above-mentioned EP pipe is placed on magnetic sheet, is washed with 1mlMES 2 times, abandon supernatant；
Configure the MES of final concentration of 0.1M before use, it includes the concentration of EDC and NHS and is 40g/L, add above two thing
After matter, it is placed on agitator abundant mixing 15 minutes, stand-by, with the resuspended magnetic bead of this liquid；
Adsorb magnetic bead with magnetic sheet, abandon supernatant, then cleaned with MES once, after absorption magnetic bead, abandon supernatant；
3) the linking of magnetic bead and albumen；
The concentration adding 50 μ L in processed good magnetic bead is the LAM polyclonal antibody of 1mg/ml and 500 μ L that pH value is 5.5
PBS, mixes, room temperature, is placed in sustained oscillation on shaking table, is incubated 2 hours；
With pH5.5, the PBS of 0.05%Tween cleans 3 times, abandons supernatant；
Add 1ml, 0.1M, 0.1%BSA Tris buffer, mix, room temperature, be placed in sustained oscillation on shaking table, be incubated 2 hours；
Add 1ml, the PBS of pH5.5,0.05%Tween, stand 2 minutes, abandon supernatant；It is repeated 3 times, add the pH=of 1ml
5.5 PBS, abandons supernatant, is repeated 3 times；When abandoning supernatant, test tube need to be placed on magnetic sheet；
Add 1ml, pH=7.4,0.05%Tween-20,0.1%BSA, 0.05%NaN3PBS, stand-by；
(4) LAM serial standards are prepared；
LAM, pH value be 7.4 PBS be diluted to 1,5,25,125,625ng/ml.
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