CN109342740A - A kind of fluorescence immune chromatography kit and preparation method thereof detecting people's pylori spiral bacilli antibody - Google Patents
A kind of fluorescence immune chromatography kit and preparation method thereof detecting people's pylori spiral bacilli antibody Download PDFInfo
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- CN109342740A CN109342740A CN201811438942.2A CN201811438942A CN109342740A CN 109342740 A CN109342740 A CN 109342740A CN 201811438942 A CN201811438942 A CN 201811438942A CN 109342740 A CN109342740 A CN 109342740A
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Abstract
The invention belongs to external diagnosis reagent immunochromatography diagnostic techniques fields, more particularly to a kind of fluorescence immune chromatography kit and preparation method thereof for detecting people's pylori spiral bacilli antibody, the kit includes support, fixed blotting membrane on the support, it is fixed on the water absorption pad of blotting membrane one end and the bonding pad of the other end, the other end of bonding pad is fixed with sample pad, and the sample pad other end is fixed with blood filter membrane；The detection line of coating Caga A, urease and vacuolate cytotoxin recombination antigen mixture, and the control line of coating sheep anti-mouse igg are provided on the blotting membrane；The bonding pad spray has the fluorescent latex microballoon of coupling mouse anti-human igg.The kit is realized using the fluorescent characteristic of indirect method principle combination fluorescent latex microballoon and carries out quick, sensitive quantitative detection to pylori spiral bacilli antibody using the method for fluorescence immune chromatography.
Description
Technical field
The invention belongs to external diagnosis reagent immunochromatography diagnostic techniques fields, and in particular to a kind of detection people pylorus spiral
Fluorescence immune chromatography kit of bacillus antibody and preparation method thereof.
Background technique
Helicobacter pylori (Helicobacter pylori, HP) is that one kind colonizes in stomach and duodenal gram
Negative microaerofil bacterium, infection is very universal, and global general population infection rate is more than 50%.Almost all of helicobacter pylorus
Histologically there is chronic active inflammatory reaction in bacterium the infected, HP gastritis is clearly " a kind of by newest Consensus of experts
Infect (infection) property disease ", but wherein about 70% or more the infected is asymptomatic, and other can lead to chronic gastritis, peptic ulcer
Deng common sympton includes epigastric sense of discomfort and pain, flatulence, anorexia, Nausea and vomiting and dark color or tar color excrement
Deng.Research shows that helicobacter pylori is the main pathogenic of gastritis, peptic ulcer, and with functional dyspepsia FD, stomach
Mucous membrane associated lymphoid tissue (MALT) lymthoma, the generation of gastric cancer are closely related, World Health Organization's international cancer research aircraft
Structure has been included in a kind of carcinogen.It is pointed out in international guidelines in relation to helicobacter pylori infections, eradicating helicobacter pylori can
It substantially reduces stomach and dudenal disease includes the disease incidence of gastric cancer, and can be in the following neopathy for reducing helicobacter pylori infections
Example.
The diagnostic methods of helicobacter pylori infections has non-invasive to be divided into two major classes according to materials: invasive detection method and
Noninvasive detection method.The former, which refers to, relies on the detection method that gastroscope is drawn materials, including histology (such as HE dyeing,
Warthin-Starry silver staining, improvement Giemsa dyeing, Toluidine blue staining, acridine orange dyeing, immunohistochemical staining
Deng), Bacteria Culture and rapid urease test (RUT) etc.；The latter then include serology (antibody) detection, Stool antigen test and
Urea breath test (UBT) etc..Different diagnostic methods has respective benefit and limitation.
The detection kit of existing helicobacter pylori there are preparation methods it is complicated, time-consuming, expensive the problems such as.
Summary of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, a kind of detection people's pylori spiral bacilli antibody is provided
Fluorescence immune chromatography kit and preparation method thereof.
The present invention is achieved by the following technical solutions:
A kind of fluorescence immune chromatography kit detecting people's pylori spiral bacilli antibody, including support (1), are fixed on branch
The blotting membrane (2) on object (1) is held, the water absorption pad (3) of blotting membrane (2) one end and the bonding pad (4) of the other end, bonding pad are fixed on
(4) the other end is fixed with sample pad (5), and sample pad (5) other end is fixed with blood filter membrane (6)；It is arranged on the blotting membrane (2)
There are the detection line (21) of coating Caga A, urease and vacuolate cytotoxin (vacA) recombination antigen mixture, and coating sheep
The control line (22) of anti-mouse IgG；Bonding pad (4) spray has the fluorescent latex microballoon of coupling mouse anti-human igg.
Preferably, the support (1) is plastic plate.
Preferably, the blotting membrane (2) is nitrocellulose filter.
Preferably, on the detection line (21) coated Caga A, urease and vacuolate cytotoxin recombinant antigen matter
Amount is than being (2-3): (0.5-1.5): (0.5-1.5).
Preferably, the fluorescent latex microballoon is the polystyrene latex microballoon in conjunction with fluorescent marker probe, can be by light
Electric multiplier tube directly detects excitation fluorescence signal, has higher temperature signal-to-noise ratio, higher signal detection amount and detection sensitivity.
A kind of preparation method of the fluorescence immune chromatography kit of detection people's pylori spiral bacilli antibody, including with
Lower step:
(1) blotting membrane (2) for having detection line (21), control line (22) is drawn in preparation, and dry 0.5-1.5 is small under 37 DEG C of environment
When；
(2) preparation spray has the bonding pad (4) of coupling mouse anti-human igg fluorescent latex microballoon, the dry 2-4 under 37 DEG C of environment
Hour；
(3) water absorption pad is arranged in blotting membrane (2) one end in the blotting membrane (2) that step (1) preparation is placed on support (1),
Sample pad (5) are arranged in bonding pad (4) other end in the bonding pad (4) of other end setting steps (2) preparation, another in sample pad (5)
Blood filter membrane (6) are arranged in one end.
Preferably, detection line (21) described in step (1) and control line (22) need continuous and even thickness.
Preferably, step (2) the preparation spray has the bonding pad of coupling mouse anti-human igg fluorescent latex microballoon to specifically include
Following sequential steps:
(21) take fluorescent latex microballoon that revolving speed 10000-15000rpm after just washing lotion ultrasound mixing is added, centrifugation is no less than
15min abandons supernatant；
(22) activating solution 1 and activating solution 2 are sequentially added, 10-30min is activated, activation condition is 35-40 DEG C, 100-
150rpm；Revolving speed 10000-15000rpm after activation, centrifugation are no less than 15min, abandon supernatant；
(23) plus coupling buffer ultrasound to clear rear centrifugation is no less than 15min, abandons supernatant, obtains coupling liquid；
(24) mouse anti-human igg is added and coupling liquid mixes, coupling, centrifugation is no less than 15min after reaction；
(25) it seals up and closes fluid-tight and close, centrifugation is no less than 15min after closing, abandons supernatant；
(26) plus whole washing lotion ultrasound to clear rear centrifugation is no less than 15min, abandons supernatant；
(27) add microballoon diluted constant volume；
(28) it will be sprayed on glass fibre element film after the microballoon diluted of the microballoon after constant volume, spray volume 8-12
μL/cm；
Wherein, first washing lotion be 1.95g/L morpholino b acid and 0.5 ‰ Tween-20, NaOH tune pH to 5.5；
Activating solution 1 is the N- hydroxy thiosuccinimide sodium salt that first washing lotion is prepared, concentration 20mg/mL；
Activating solution 2 is 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride that first washing lotion is prepared, concentration
20mg/mL；
Coupling buffer be 1.95g/L morpholino b acid and 0.5 ‰ Tween-20, NaOH tune pH to 7.0；
Confining liquid is the bovine serum albumin(BSA) of 5g/L, the borate buffer of 2 ‰ Tween-20 and 0.2mol/L；
Whole washing lotion is the ox blood of the Trisbase of 6g/L, 0.5 ‰ Tween-20,0.3 ‰ Proclin 300 and 5g/L
Pure albumen adjusts pH to 8.0；
Microballoon dilution be 20% trehalose, 1%S9,0.5% bovine serum albumin(BSA), 0.5 ‰ Tween-20 and 10mM
Trisbase adjusts pH to 8.0.
Preferably, step (22) the fluorescent microsphere activation process need to be carried out in the dark.
Preferably, the mass ratio of step (24) the mouse anti-human igg and coupling liquid is (0.5-1.0): (3-6), coupling side
Method is 37 DEG C, 100-150rpm coupling is no less than 2 hours.
Preferably, step (25) enclosure method is plus 37 DEG C of confining liquid, 150rpm closing activity group are no less than 30-
45min。
Preferably, the ultrasound is completely dispersed microballoon and its coupled antibody, the limpid light transmission of solution in centrifuge tube.
Preferably, step (28) microballoon to gush out answers fog-like be dispersed on glass fibre element film.
Design principle of the invention is: reagent is added when in use, by sample to be tested in helicobacter pylori detection kit
At the sample point sample of box (blood filter membrane), blood filter membrane can be except the blood clotting in serum deprivation, when sample passes through bonding pad, on bonding pad
Microballoon coupled antibody mouse anti-human igg human IgG antibody, microballoon can pass through the detection line of blotting membrane in indiscriminate stripped serum
When, the HP antibody in human IgG antibody adsorbed thereon can be detected coated specificity HP recombinant antigen on survey line and catch, Zhi Houtong
When crossing control line, the mouse anti-human IgG antibodies of microballoon absorption are detected the coated sheep anti-mouse igg antibody capture of survey line, therefore can quantify
Detect the pylori antigen in sample.
The beneficial effects of the present invention are:
Helicobacter pylori detection kit of the present invention, it is special using the fluorescence of indirect method principle combination fluorescent latex microballoon
Property the method that realizes using fluorescence immune chromatography quick, sensitive quantitative detection is carried out to pylori spiral bacilli antibody.This hair
The bright helicobacter pylori detection preparation method of kit, simple and quick, convenient for control, cost is relatively low.
Detailed description of the invention
Fig. 1 is the schematic diagram of the fluorescence immune chromatography kit of present invention detection people's pylori spiral bacilli antibody；
Fig. 2 is for the luminous value ratio (T/C value) of detection line (T line) and control line (C line) in the embodiment of the present invention 3 and accordingly
The standard curve of standard concentration.
Appended drawing reference:
1, support；2, blotting membrane；3, water absorption pad；4, bonding pad；5, sample pad；6, blood filter membrane.
Specific embodiment
To be best understood from the present invention, below with reference to examples and drawings, the invention will be further described, following embodiment
It is only that the present invention will be described rather than is limited to it.
Embodiment 1
The present embodiment helicobacter pylori detection kit is arranged as shown in Figure 1, including support 1 in support 1
On blotting membrane 2, setting is water absorption pad 3 in 2 one end of blotting membrane, and the corresponding other end is bonding pad 4, is arranged another in bonding pad 4
End is sample pad 5, and setting is blood filter membrane 6 in 5 other end of sample pad.Wherein, support 1 selects plastic plate, and blotting membrane 2 selects nitre
Acid cellulose film.Be provided with detection line 21 and control line 22 on nitrocellulose membrane, detection line 21 be coated with Caga A, urease and
Vacuolate cytotoxin (vacA) recombination antigen mixture；Control line 22 is coated with sheep anti-mouse igg.4 spray of bonding pad has coupling mouse
The fluorescent latex microballoon of anti-human igg.Coated Caga A, urease and cell blown-out shot toxin base in nitrocellulose membrane detection line 21
Because the mixing mass ratio of (vacA) recombinant antigen is (2-3): (0.5-1.5): (0.5-1.5).
The present embodiment helicobacter pylori detection preparation method of kit, specific steps are as follows:
(1) nitrocellulose film preparation:
It is 2- that Caga A, urease and cell blown-out shot toxin (vacA) recombinant antigen, which are diluted to concentration with phosphate buffer,
The hybrid antigen of 3mg/mL, 0.5-1.5mg/mL, 0.5-1.5mg/mL.Sheep anti-mouse igg is diluted to concentration with phosphate buffer
For 1mg/mL.Using spray film lining instrument by HP recombinant antigen (Caga A, urease and vacA recombination antigen mixture) and goat-anti
Mouse IgG crosses on nitrocellulose filter respectively, 37 DEG C drying 1 hour, it is spare.
(2) prepared by bonding pad:
1. solution is prepared
A: morpholino b acid that first washing lotion is 1.95g/L and 5/10000ths Tween-20, sodium hydroxide tune pH to 5.5.
B: activating solution 1 is the diluted N- hydroxy thiosuccinimide sodium salt of first washing lotion, concentration 20mg/mL；Activating solution 2 is
First washing lotion diluted 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride, concentration 20mg/mL.
C: coupling liquid be 1.95g/L morpholino b acid and 0.5 ‰ Tween-20, sodium hydroxide tune PH to 7.0.
D: confining liquid is the bovine serum albumin(BSA) of 5g/L, the borate buffer of 2 ‰ Tween-20 and 0.2mol/L.
E: whole washing lotion is the ox of the Trisbase of 6g/L, 0.5 ‰ Tween-20,0.3 ‰ Proclin 300 and 5g/L
Seralbumin adjusts pH value 8.0.
F: microballoon dilution be 20% trehalose, 1%S9,0.5% bovine serum albumin(BSA), 0.5 ‰ Tween-20 with
10mM Trisbase adjusts PH to 8.0.
2. fluorescent latex microballoon coupled antibody
A: 15000rpm centrifugation 15min after taking fluorescent latex microballoon that just washing lotion ultrasound mixing is added abandons supernatant.
B: sequentially adding activating solution 1 and activating solution 2, activates 15min in 37 DEG C of 150rpm；15000rpm is centrifuged after activation
15min abandons supernatant.
C: add coupling liquid ultrasound to clear rear 15000rpm centrifugation 15min, abandon supernatant.
D: being added mouse anti-human igg and coupling liquid mixes, and in being coupled 2 hours on 37 DEG C of 150rpm shaking tables, is centrifuged 15min, abandons
Supernatant.
E: add the active group of 500uL confining liquid closing microballoon, close 30min, 15000rpm on 37 DEG C of 150rpm shaking tables
It is centrifuged 15min, abandons supernatant.
F: add whole washing lotion ultrasound to clear rear centrifugation 15min, abandon supernatant.
G: microballoon diluted is added to be settled to 1 milliliter.
3. prepared by bonding pad
It on the glass fibre element film for spraying 7 mm in width, will be sprayed after 10 times of the microballoon dilution dilutions of microballoon after constant volume
Volume processed is 12 μ L/cm.37 DEG C drying 3 hours, it is spare.
(3) reagent strip assembles
The nitrocellulose filter that step (1) preparation is placed on plastic plate is water absorption pad in nitrocellulose filter one end, right
Answering the other end is bonding pad, and setting is sample pad in the bonding pad other end, and setting is blood filter membrane in the sample pad other end.
Embodiment 2
The present embodiment helicobacter pylori detection kit, unlike the first embodiment, in microballoon coupled antibody process
In, in being coupled 1 hour on 37 DEG C of 100-150rpm shaking tables, the work for directly adding 500uL confining liquid closing microballoon is not centrifuged after coupling
Property group closes 30min on 37 DEG C of 150rpm shaking tables.Other are the same as embodiment 1.Embodiment 1 and embodiment 2 detect identical sample
This, data are shown in Table 1.
Table 1
Coefficient of variation CV < 15% of detectable concentration when embodiment 1 and embodiment 2 detect identical sample, embodiment 1 and reality
2 two kinds of reagent strip testing results of example are applied without significant difference.
Embodiment 3
By pylori spiral bacilli antibody be diluted to 1.56ug/L, 3.13ug/L, 6.25ug/L, 12.5ug/L, 25ug/L,
Totally 6 concentration gradients while preparing blank solution as standard solution to 50ug/L.
6 standard items are taken respectively and each 80 microlitres of the blank solution sample application zones (sample pad) for being added to reagent strip, after 15min
Being inserted into HitFia- must (must Ou Han biotechnology (Hefei) Co., Ltd) reading, each standard on Ou Han fluorescence immunity analyzer
Product examine is surveyed 3 times.The luminous value and T/C value for reading detection line (T line) and control line (C line), are shown in Table 2.Calculate T/C value and corresponding mark
The concentration relationship of quasi- product draws standard curve, sees Fig. 2.Standard curve regression equation is shown in Table 3.Detection limit can reach 0.5ug/L.
Table 2
3 four parameter Logistic curve matching of table
Embodiment 4
36 random clinical samples are taken, helicobacter pylori is resisted using kit prepared by the embodiment of the present invention 1
Body is detected.Detection method are as follows: 80 microlitres of samples to be tested instill in reagent strip sample application zone (sample pad), are inserted into after 15min
HitFia- must (must Ou Han biotechnology (Hefei) Co., Ltd) reading, each standard product examine on Ou Han fluorescence immunity analyzer
It surveys 2 times.Luminous value, the T/C value for reading detection line (T line) and control line (C line) substitute into the T/C value of sample in standard curve,
Concentration value is calculated, the results are shown in Table 4.The sensitivity of the present embodiment reagent strip and precision are shown in Table 5.
Table 4
| Clinical sample | T line | C line | T/C value | Concentration (ug/L) |
| 1 | 9061588 | 26882920 | 0.3371 | 2.705698 |
| 2 | 4994028 | 23538712 | 0.2122 | 1.689066 |
| 3 | 45583015 | 19509403 | 2.3365 | 15.162556 |
| 4 | 11802745 | 21893044 | 0.5391 | 4.203843 |
| 5 | 18458660 | 23788326 | 0.776 | 5.831909 |
| 6 | 100258949 | 23906532 | 4.1938 | 25.008855 |
| 7 | 18279147 | 30951452 | 0.5906 | 4.567257 |
| 8 | 5492716 | 24792292 | 0.2215 | 1.768491 |
| 9 | 91866619 | 22031306 | 4.1698 | 24.886184 |
| 10 | 12072586 | 24707389 | 0.4886 | 3.841189 |
| 11 | 51319854 | 17477930 | 2.9363 | 18.435057 |
| 12 | 51220547 | 16113473 | 3.1787 | 19.728712 |
| 13 | 5222179 | 19047003 | 0.2742 | 2.205924 |
| 14 | 18215609 | 29574256 | 0.6159 | 4.743642 |
| 15 | 19353300 | 17600183 | 1.0996 | 7.920355 |
| 16 | 8156382 | 24811035 | 0.3287 | 2.640127 |
| 17 | 14644277 | 23667593 | 0.6187 | 4.763081 |
| 18 | 28539918 | 22067688 | 1.2933 | 9.118542 |
| 19 | 6764029 | 27531361 | 0.2457 | 1.971845 |
| 20 | 6605618 | 21368035 | 0.3091 | 2.485797 |
| 21 | 21021202 | 26887948 | 0.7818 | 5.870533 |
| 22 | 16358589 | 20769579 | 0.7876 | 5.909105 |
| 23 | 7193799 | 32566038 | 0.2209 | 1.763389 |
| 24 | 5533710 | 20988091 | 0.2637 | 2.12031 |
| 25 | 25105438 | 20151398 | 1.2458 | 8.827686 |
| 26 | 48177466 | 20517652 | 2.3481 | 15.226955 |
| 27 | 13881880 | 22176175 | 0.626 | 4.813686 |
| 28 | 7040884 | 31932716 | 0.2205 | 1.759986 |
| 29 | 18220062 | 22778997 | 0.7999 | 5.990741 |
| 30 | 15222162 | 22975404 | 0.6625 | 5.065139 |
| 31 | 4861388 | 19546091 | 0.2487 | 1.996745 |
| 32 | 8000173 | 27909558 | 0.2866 | 2.306158 |
| 33 | 15559180 | 20174699 | 0.7712 | 5.799906 |
| 34 | 5466543 | 22621667 | 0.2417 | 1.938543 |
| 35 | 7460661 | 21102269 | 0.3535 | 2.832793 |
| 36 | 13380333 | 26930125 | 0.4969 | 3.901256 |
Table 5
It can be seen that this method can direct quantitative measure pylori spiral bacilli antibody concentration, it is easy to operate quickly, 15min
Can be obtained as a result, and low in cost, high sensitivity, high specificity.
Embodiment described above is only that preferred embodiments of the present invention will be described, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (9)
1. a kind of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody, including support (1), are fixed on support
Blotting membrane (2) on object (1) is fixed on the water absorption pad (3) of blotting membrane (2) one end and the bonding pad (4) of the other end, bonding pad
(4) the other end is fixed with sample pad (5), and sample pad (5) other end is fixed with blood filter membrane (6)；It is characterized by: the trace
The detection line (21) of coating Caga A, urease and vacuolate cytotoxin recombination antigen mixture, and packet are provided on film (2)
By the control line (22) of sheep anti-mouse igg；Bonding pad (4) spray has the fluorescent latex microballoon of coupling mouse anti-human igg.
2. a kind of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 1, special
Sign is: the support (1) is plastic plate.
3. a kind of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 1, special
Sign is: the blotting membrane (2) is nitrocellulose filter.
4. a kind of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 1, special
Sign is: the mass ratio of coated Caga A, urease and vacuolate cytotoxin recombinant antigen are (2- on the detection line (21)
3): (0.5-1.5): (0.5-1.5).
5. a kind of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 1, special
Sign is: the fluorescent latex microballoon is the polystyrene latex microballoon in conjunction with fluorescent marker probe.
6. a kind of fluorescence immune chromatography kit of the described in any item detection people's pylori spiral bacilli antibodies of claim 1 ~ 5
Preparation method, which comprises the following steps:
(1) blotting membrane (2) for having detection line (21), control line (22) is drawn in preparation, 0.5-1.5 hours dry under 37 DEG C of environment；
(2) preparation spray has the bonding pad (4) of coupling mouse anti-human igg fluorescent latex microballoon, and dry 2-4 is small under 37 DEG C of environment
When；
(3) water absorption pad is arranged in blotting membrane (2) one end in the blotting membrane (2) that step (1) preparation is placed on support (1), another
Sample pad (5) are arranged in bonding pad (4) other end, in sample pad (5) other end in the bonding pad (4) for holding setting steps (2) preparation
It is arranged blood filter membrane (6).
7. a kind of preparation of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 6
Method, it is characterised in that: detection line (21) described in step (1) and control line (22) need continuous and even thickness.
8. a kind of preparation of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 6
Method, which is characterized in that step (2) the preparation spray has the bonding pad of coupling mouse anti-human igg fluorescent latex microballoon specifically to wrap
Include following sequential steps:
(21) take fluorescent latex microballoon that revolving speed 10000-15000rpm after just washing lotion ultrasound mixing is added, centrifugation is no less than 15min,
Abandon supernatant；
(22) activating solution 1 and activating solution 2 are sequentially added, 10-30min is activated, activation condition is 35-40 DEG C, 100-150rpm；It is living
Revolving speed 10000-15000rpm after change, centrifugation are no less than 15min, abandon supernatant；
(23) plus coupling buffer ultrasound to clear rear centrifugation is no less than 15min, abandons supernatant, obtains coupling liquid；
(24) mouse anti-human igg is added and coupling liquid mixes, coupling, centrifugation is no less than 15min after reaction；
(25) it seals up and closes fluid-tight and close, centrifugation is no less than 15min after closing, abandons supernatant；
(26) plus whole washing lotion ultrasound to clear rear centrifugation is no less than 15min, abandons supernatant；
(27) add microballoon diluted constant volume；
(28) it will be sprayed on glass fibre element film after the microballoon diluted of the microballoon after constant volume, spray volume is 8-12 μ L/
cm；
Wherein, first washing lotion be 1.95g/L morpholino b acid and 0.5 ‰ Tween-20, NaOH tune pH to 5.5；
Activating solution 1 is the N- hydroxy thiosuccinimide sodium salt that first washing lotion is prepared, concentration 20mg/mL；
Activating solution 2 is 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride that first washing lotion is prepared, concentration 20mg/mL；
Coupling buffer be 1.95g/L morpholino b acid and 0.5 ‰ Tween-20, NaOH tune pH to 7.0；
Confining liquid is the bovine serum albumin(BSA) of 5g/L, the borate buffer of 2 ‰ Tween-20 and 0.2mol/L；
Whole washing lotion is that Trisbase, 0.5 ‰ Tween-20, the ox blood of 0.3 ‰ Proclin 300 and 5g/L of 6g/L is pure
Albumen adjusts pH to 8.0；
Microballoon dilution be 20% trehalose, 1%S9,0.5% bovine serum albumin(BSA), 0.5 ‰ Tween-20 and 10mM
Trisbase adjusts pH to 8.0.
9. a kind of preparation of fluorescence immune chromatography kit for detecting people's pylori spiral bacilli antibody according to claim 8
Method, it is characterised in that: step (22) the fluorescent microsphere activation process need to be carried out in the dark；
Step (24) the mouse anti-human igg and coupling liquid mass ratio be (0.5-1.0): (3-6), coupling method be 37 DEG C,
100-150rpm coupling no less than 2 hours；
Step (25) enclosure method is to add 37 DEG C of confining liquid, 100-150rpm closing activity group 30-45min；
The ultrasound is completely dispersed microballoon and its coupled antibody, the limpid light transmission of solution in centrifuge tube；
Step (28) microballoon to gush out answers fog-like be dispersed on glass fibre element film.
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