CN108956989A - A kind of classification of helicobacter pylori detection detection reagent card and preparation method thereof - Google Patents
A kind of classification of helicobacter pylori detection detection reagent card and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of classification of helicobacter pylori detection detection reagent cards, it includes sample pad, quantum pad, nitrocellulose filter, blotting paper and liner plate, the antigenic compound of quantum dot microsphere label is coated on quantum pad, antigenic compound includes Urease antigen, CagA antigen, VacA antigen and mouse IgG；The first detection line, the second detection line, third detection line and nature controlling line are provided on nitrocellulose filter, VacA antigen is coated in first detection line, it is coated with CagA antigen in second detection line, Urease antigen is coated in third detection line, is coated with sheep anti-mouse igg on nature controlling line.Provided by the present invention for detecting the detection reagent card of classification of helicobacter pylori, can fast and accurately obtain as a result, and can judge the infection type of helicobacter pylori according to testing result, and then provide assistance in diagnosis for related disease clinically.
Description
Technical field
The invention belongs to quantum dot immune fluorescent chromatographic technique fields in Medical Immunology, and in particular to a kind of helicobacter pylorus
Bacterium parting detection detection reagent card and preparation method thereof.
Background technique
Epidemiological study shows that helicobacter pylori (HP) infection rate is very high, and the people in the whole world about 50% carries
HP is confirmed as the most important virulence factor of the disease of stomach such as gastric ulcer, chronic gastritis and gastric cancer by international cancer tissue.Hair
The height and social and economic level of sick rate, densely populated degree, public health condition and water source supply have compared with close relationship.
Epidemiological study shows that HP infection mostly occurs in the Childhood, and when lacking antibiotic treatment, HP infection is usually adjoint
Throughout one's life；The genotype of childhood infection bacterial strain is not changed by environmental transport and transfer, often consistent with the HP genotype of parent's infection.In addition,
HP infection is also widest chronic bacterial infection in adult, and infection rate increases with the age and risen.Chinese general population
The investigation of HP infection rate shows: 21 save 52 areas, add up detection number 25209, HP infection rate from 34.52%~80.55%,
Most area crowd infection rates are 50% or so, average rate 58.07%, high incidence area of gastric cancer crowd's HP infection rate total water
It is flat to be higher than the area Di Fa crowd, the infection age earlier than developed country 20 years or so, 20 years old~40 years old infection rate be 45.4%~
63.6%, 70 years old or more up to 78.9%.
Research confirms that HP can induce the change of atrophic inflammation, and then gastrin secretion increases, Transitional cell carcinomas
(COX-2) rise with prostaglandin (PGE) expression quantity, cause gastric mucosal cell form and function to change, lead to chronic gastritis, stomach
The generation of the enterogastric diseases such as ulcer, duodenal ulcer, gastric cancer and gastric lymphoma of mucosa-associated lymphoid tissue.HP infection significantly increases hair
Peptic ulcer disease may occur for the risk of raw duodenum and gastric ulcer, about 1/6 infected with Helicobacter pylori；HP infection
So that the danger to get a cancer of the stomach is increased 2.7~12 times, has 5% Type B atrophic gastritis patient that can develop as gastric cancer.With research
Go deep into, it has been found that HP infect in non-digestive tract disease, such as hypoferric anemia, Idiopathic Thrombocytopenic Purpura, liver and gallbladder
It similarly plays a significant role in the occurrence and development of pipe disease, cardiovascular disease, diabetes, Chronic Obstructive Pulmonary Disease etc..
Helicobacter pylori is Gram-negative bacteria, and thallus is twist bent, therefore claims helicobacter pylori.Helicobacter pylori is raw
It is stored in stomach and duodenal each region, the chronic inflammation that it can cause stomach lining slight, even lead to stomach and 12 fingers
Enterelcosis and gastric cancer.Helicobacter pylori is the bacterium that a large amount of uremic enzymes can be uniquely generated in people's stomach, therefore can be by detecting uremic
Enzyme infects to diagnose HP.1994, International Agency for Research on Cancer by HP infection is set to I class carcinogen, can carcinogenic HP mainly contain
Two kinds of cytotoxins: one is cytotoxin-associated protein CagA, and one is vacuolating cytotoxin VacA.According to whether expression
CagA and VacA divides HP for two types: I type is that (CagA and VacA are expressed, or expression for pathogenic stronger toxigenic bacterium strain
It is one of), II type is the weaker not toxigenic bacterium strain of toxicity (CagA and VacA are not expressed).
Since nineteen eighty-three takes biopsy specimen to be separately cultured successfully by gastroscope, the diagnosis of HP infection is had been developed and has been permitted
Multi-method, such as immunology, bacteriology, pathology, serology, tagging, molecular biology.It can from collection of specimens angle
To be divided into invasive and Noninvasive two major classes.Invasive method refers mainly to take the side of biopsy specimen inspection by gastroscope
Method, being separately cultured with direct smear, rapid urease test, fluorescent PCR (TaqMan probe) etc. including bacterium.Noninvasive
Method refer mainly to not by gastroscope take biopsy specimen diagnose HP method, including tracer method, immunological method (antigen,
Antibody test) etc..Detection method is although a variety of, but it is easy, quickly can also detection and genotyping it is very little.
Quantum dot microsphere has the preferable biofacies same sex, and wherein quantum dot vector can be polystyrene, poly- methyl-prop
E pioic acid methyl ester, polybutadiene etc., and ideal carrier for fluorescent material is polystyrene at present, have be easily-synthesized, surface
The advantages that easily modifying organo-functional group and high reactivity.The quantum dot polystyrene microsphere of formation is hydrophobic, it is hard, can not biology
It degrades, not by common solvent dissolution or swelling, has large specific surface area, strong adsorption, coagulation big and surface respond
The characteristics such as strong.
Quantum dot fluorescence microballoon has higher fluorescence intensity, and it is quick, highly sensitive, inexpensive etc. excellent that this has product testing
Point is possibly realized.Quantum dot microsphere is a large amount of quantum dots to be contained or are adsorbed onto the microballoon that inside microballoon or surface is formed.It is mentioned
Quantum dot is loaded in inside microballoon while the fluorescence intensity of high single microballoon, moreover it is possible to improve the stability of microballoon, such as anti-drift
Bai Xing, thermal stability, chemical stability etc..Functional group or polyelectrolyte etc. are modified in microsphere surface simultaneously, is convenient for and biology
Macromolecular is combined by modes such as chemistry, electrostatic, hydrogen bonds, can be widely applied to each field of biology.
Summary of the invention
Technical problem to be solved by the invention is to provide it is a kind of work simplicity, rapid reaction, high specificity helicobacter pylorus
Bacterium parting detection detection reagent card and preparation method thereof.
In order to solve the above technical problems, the present invention adopts the following technical scheme that:
It is an object of the present invention to provide a kind of classification of helicobacter pylori detection detection reagent cards comprising sample
Pad, quantum pad, nitrocellulose filter, blotting paper and liner plate are coated with the antigen of quantum dot microsphere label on the quantum pad
Compound, the antigenic compound include Urease antigen, CagA antigen, VacA antigen and mouse IgG；The cellulose nitrate
Lateral side where the blotting paper is disposed with the first detection line, the second detection from where the sample pad on plain film
Line, third detection line and nature controlling line are coated with VacA antigen in first detection line, are coated in second detection line
There is CagA antigen, Urease antigen is coated in the third detection line, is coated with sheep anti-mouse igg on the nature controlling line.
Preferably, the quantum dot microsphere includes quantum dot polystyrene microsphere, is deposited on the quantum dot polyphenyl
One or more layers polyelectrolyte layer of ethylene microsphere surface, the polyelectrolyte layer include being formed by polyallylamine hydrochloride
Internal layer and the outer layer for being deposited on the internal layer surface formed by kayexalate.
Specifically, the quantum dot microsphere is made by the steps to obtain:
(1) the quantum dot polystyrene microsphere is dispersed in water, polyallylamine hydrochloride solution is added, shaking table is incubated
Change the internal layer that 30~60min forms the polyelectrolyte layer, centrifugation removes extra polyallylamine hydrochloride；
(2) step (1) the quantum dot polystyrene microsphere obtained for being formed with the internal layer is dispersed in water, is added
Kayexalate solution, shaking table hatch the outer layer that 30~60min forms the polyelectrolyte layer, and centrifugation removal is extra
Kayexalate；
(3) step (1) and step (2) are selectively repeated, the quantum dot microsphere is obtained.
In the present invention, the quantum dot polystyrene microsphere can be micro- using the quantum dot polystyrene of commercially available acquisition
Ball, or using quantum dot polystyrene microsphere made from conventional method in that art.
More specifically, in step (1), the throwing of the quantum dot polystyrene microsphere and the polyallylamine hydrochloride
Material mass ratio is 1:60~70；In step (2), the formation have the quantum dot polystyrene microsphere of the internal layer with it is described
Kayexalate feed intake mass ratio be 1:60~70.
More specifically, it is 1~3mg/mL that the quantum dot polystyrene microsphere, which is diluted with water to concentration, in step (1).
More specifically, the concentration of the polyallylamine hydrochloride solution is 1~3mg/mL in step (1).
More specifically, the quantum dot polystyrene microsphere for being formed with the internal layer is diluted with water to concentration in step (2)
For 1~3mg/mL.
More specifically, the concentration of the kayexalate solution is 1~3mg/mL in step (2).
Preferably, the quantum dot in the quantum dot microsphere is CdSe/CdS and/or CdSe/ZnS.
Preferably, Urease antigen described in the antigenic compound, the CagA antigen, the VacA are anti-
The mass ratio that feeds intake of former and described mouse IgG is 1:0.9~1,1:0.9~1,1:0.9~1,1.
Preferably, the mass ratio that feeds intake of the quantum dot microsphere and the antigenic compound is 1:0.6~1.
Preferably, the quantum pad is to pass through pretreated quantum pad, the pretreated method are as follows: by quantum pad
It is immersed in quantum pad optimization buffer, then dries and control humidity below 30%, wherein the quantum pad optimization is slow
Fliud flushing include concentration be 45~55mmol/L trishydroxymethylaminomethane, mass percentage concentration be 0.8~1.2% cow's serum
Sucrose that polysorbas20 that albumin, mass percentage concentration are 0.4~0.6%, mass percentage concentration are 1.8~2.2%, quality hundred
Divide the purified water of the concentration proclin300 for being 0.08~0.12% and surplus.
It is a further object to provide the systems of the classification of helicobacter pylori detection detection reagent card described in one kind
Preparation Method includes the following steps:
(1) quantum dot microsphere is dispersed in phosphate buffer, is then added the antigenic compound, 10
~40 DEG C of shaking tables hatch 1.5~2.5h, add quantum dot microsphere confining liquid, hatch 2~3h, and centrifugal filtration obtains the amount
The antigenic compound of son point microballoon label, wherein the quantum dot microsphere confining liquid includes that mass percentage concentration is 8~12%
Bovine serum albumin(BSA), mass percentage concentration be 0.04~0.06% NaN3And the purified water of surplus；
(2) by the antigenic compound quantum dot dilution buffer of quantum dot microsphere label described made from step (1)
It is diluted to 0.4~0.6 μm of ol/L, is then sprayed on the amount of 0.8~1.2 μ L/cm on the quantum pad, it is dry that coating is made
The quantum pad for the antigenic compound for thering is quantum dot microsphere to mark, wherein the quantum dot dilution buffer includes that concentration is 90
The trishydroxymethylaminomethane of~110mmol/L, the bovine serum albumin(BSA) that mass percentage concentration is 0.8~1.2%, quality percentage
Sucrose that polysorbas20 that concentration is 0.4~0.6%, mass percentage concentration are 1.8~2.2%, mass percentage concentration be 0.08~
0.12% proclin300 and the purified water of surplus；
(3) nitrocellulose filter is pasted on the liner plate, by the VacA antigen, the CagA
Antigen and the Urease antigen are diluted to 0.8~1.2mg/m L with die buffer respectively, by the sheep anti-mouse igg
It is diluted to 0.6~1mg/m L with die buffer, is then sprayed on the nitric acid fibre with the amount of 0.8~1.2 μ L/cm respectively
Plain film is tieed up, it is dry that the first detection line, second detection line, the third detection line and the Quality Control is made
Line, wherein the die buffer includes that concentration is 0.01~0.03mol/L, the PBS buffer solution of pH7~7.4, quality hundred
Ethylenediamine tetra-acetic acid that trehalose that point concentration is 0.8~1.2%, concentration are 8~12mmol/L, mass percentage concentration 0.08
~0.12% proclin300；
It (4) will treated is pasted with the liner plate of nitrocellulose filter, sample pad, after step (2) processing through step (3)
Quantum pad, blotting paper assembled to obtain the detection reagent card.
Preferably, the preparation method further includes carrying out pretreated step to the sample pad: sample pad is soaked
Not in sample pad optimization buffer, then dries and control humidity below 30%, wherein the sample pad optimizes buffering
Liquid include concentration be 45~55mmol/L trishydroxymethylaminomethane, mass percentage concentration be 0.4~0.6% polysorbas20,
The purified water of proclin300 and surplus that mass percentage concentration is 0.08~0.12%.
Third object of the present invention is to provide the detection reagents described in one kind to be stuck in classification of helicobacter pylori detection
Using.
Preferably, it is detected using iSIA01 fluorescence immunity analyzer.
Technical principle of the invention is: when the test serum for containing substance to be detected is added in sample pad, serum passes through rainbow
Suction effect forms solid phase antigen-antibody-labelled antigen compound after flowing through quantum pad and nitrocellulose filter, opens from point sample
The signal value for reading detection line and nature controlling line after beginning 15 minutes by analyzer, sentences respectively according to 3 respective reference values of antibody
The yin and yang attribute of disconnected Urease antibody, CagA antibody and VacA antibody.
Due to the implementation of above technical scheme, the invention has the following advantages over the prior art:
Provided by the present invention for detecting the detection reagent card of classification of helicobacter pylori, can fast and accurately be tied
Fruit.The present invention is using Urease antibody, CagA antibody and the VacA antibody in quantum dot immune fluorescent method detection serum, according to inspection
The infection type of helicobacter pylori can be judged by surveying result, and then be provided assistance in diagnosis for related disease clinically.
Detailed description of the invention
Attached drawing 1 is the structural schematic diagram of test strips of the present invention；Wherein, 1: sample pad；2: quantum pad；3: nitrocellulose
Film；4: blotting paper；5: liner plate；6: the first detection lines；7: the second detection lines；8: third detection line；9: nature controlling line.
Specific embodiment
The present invention will be further described in detail combined with specific embodiments below, but the present invention is not limited to following implementations
Example.Implementation condition used in the examples can do further adjustment according to specifically used different requirements, the implementation being not specified
Condition is the normal condition in the industry, the commercially available acquisition of reagent in the present invention.
The preparation of embodiment 1, detection reagent card
1, the optimization of sample pad 1:
1.1, material and reagent:
(1) sample pad 1: glass fibre material.
(2) sample pad optimizes buffer: trishydroxymethylaminomethane (tris), the quality percentage of 50mmol/L, pH8.2 are dense
The purified water of polysorbas20, the proclin300 that mass percentage concentration is 0.1% and surplus that degree is 0.5%.
1.2, sample pad 1 is immersed in sample pad optimization buffer, submerges 30~45min, then the dry 5h of drying room
More than, humid control is below 30%.
2, the optimization of quantum pad 2:
2.1, material and reagent
(1) quantum pad 2: glass fibre material.
(2) quantum pad optimizes buffer: trishydroxymethylaminomethane (tris), the quality percentage of 50mmol/L, pH8.2 are dense
Spending the bovine serum albumin(BSA) (BSA) for being 1%, the tween (Tween) 20 that mass percentage concentration is 0.5%, mass percentage concentration is
The purified water of 2% sucrose, the proclin300 that mass percentage concentration is 0.1% and surplus.
2.2, quantum pad 2 is immersed in quantum pad optimization buffer, submerges 30~45min, then the dry 5h of drying room
More than, humid control is below 30%.
3, the preparation of quantum dot microsphere:
3.1, material and reagent
(1) quantum dot polystyrene microsphere: source is self-control or outsourcing, quantum dot CdSe/CdS.
(2) polyallylamine hydrochloride (PAH) solution: concentration 2mg/mL, solvent are ultrapure water.
(3) kayexalate (PSS) solution: concentration 2mg/mL, solvent are ultrapure water.
3.2, preparation step:
(1) quantum dot polystyrene microsphere is dispersed in ultrapure water after purification, being diluted to concentration is 2mg/mL, takes 3mL
Quantum dot polystyrene microsphere aqueous dispersions, are added the polyallylamine hydrochloride solution of 200mL, and shaking table hatches 30min, then with
The revolving speed centrifugation of 12000r/min removes extra PAH polyelectrolyte；
(2) quantum dot polystyrene microsphere made from step (1) is dispersed in ultrapure water, being diluted to concentration is 2mg/
ML takes 3mL quantum dot polystyrene microsphere aqueous dispersions, and the kayexalate solution of 200mL, shaking table hatching is added
Then 30min removes extra PSS polyelectrolyte with the revolving speed centrifugation of 12000r/min；
(3) quantum dot polystyrene microsphere made from step (2) is dispersed in ultrapure water, being diluted to concentration is 2mg/
ML takes 3mL quantum dot polystyrene microsphere aqueous dispersions, and the polyallylamine hydrochloride solution of 200mL, shaking table hatching is added
Then 30min removes extra PAH polyelectrolyte with the revolving speed centrifugation of 12000r/min；
(4) quantum dot polystyrene microsphere made from step (3) is dispersed in ultrapure water, being diluted to concentration is 2mg/
ML takes 3mL quantum dot polystyrene microsphere aqueous dispersions, and the kayexalate solution of 200mL, shaking table hatching is added
Then 30min removes extra PSS polyelectrolyte with the revolving speed centrifugation of 12000r/min, is made and is deposited with two layers of polyelectrolyte
The quantum dot microsphere of layer, wherein every strata electrolyte layer respectively includes the internal layer formed by polyallylamine hydrochloride and by gathering
The outer layer for being deposited on internal layer surface that sodium styrene sulfonate is formed.
4, the preparation of the antigenic compound of quantum dot microsphere label:
4.1, material and reagent
(1) quantum dot microsphere made from step 3.
(2) phosphate buffer (PBS buffer solution): group becomes sodium dihydrogen phosphate and disodium hydrogen phosphate.
(3) antigenic compound: mass ratio is Urease antigen, CagA antigen, VacA antigen and the mouse IgG of 1:1:1:1.
(4) quantum dot microsphere confining liquid: mass percentage concentration be 10% bovine serum albumin(BSA) (BSA), quality percentage it is dense
The NaN that degree is 0.05%3And the purified water of surplus.
4.2,1mg quantum dot microsphere is added in 10mL phosphate buffer, ultrasonic disperse is uniform；Then 0.8mg is added
Antigenic compound, room temperature shaker be incubated for 2h, make antigen in conjunction with quantum dot microsphere；The quantum dot microsphere closing of 0.5mL is added
Liquid hatches 2.5h, is then centrifuged 30min with the speed of 12000r/min, is centrifuged 2 times.4 DEG C of preservations.
5, it is coated with the preparation of the quantum pad 2 of the antigenic compound of quantum dot microsphere label
5.1, material and reagent
(1) the quantum pad 2 after step 2 optimization.
(2) antigenic compound of the label of quantum dot microsphere made from step 4.
(3) quantum dot dilution buffer: the trishydroxymethylaminomethane (tris) of concentration 100mmol/L, pH8.2, matter
Measure the bovine serum albumin(BSA) (BSA) that percentage concentration is 1%, the tween (Tween) 20 that mass percentage concentration is 0.5%, quality hundred
The purified water of sucrose, the proclin300 that mass percentage concentration is 0.1% and surplus that point concentration is 2%.
5.2, the antigenic compound that quantum dot microsphere marks is diluted to 0.5 μm of ol/L with quantum dot dilution buffer, so
It is sprayed on quantum pad 2 with stroke film gold spraying instrument with the amount of 1 μ L/cm afterwards, dry 0.5h.
6, on nitrocellulose filter 3 detection line and nature controlling line 9 preparation
6.1, material and reagent
(1) nitrocellulose filter 3.
(2) liner plate 5:PVC plate.
(3) VacA antigen.
(4) CagA antigen.
(5) Urease antigen.
(6) sheep anti-mouse igg.
(7) die buffer: concentration is 0.01~0.03mol/L, the PBS buffer solution of pH7.2, mass percentage concentration are
1% trehalose, the ethylenediamine tetra-acetic acid that concentration is 10mmol/L (EDTA), mass percentage concentration are 0.1%
proclin300。
6.2, nitrocellulose filter 3 is pasted on liner plate 5, VacA antigen, CagA antigen and Urease antigen is distinguished
It is diluted to 1mg/mL with die buffer, sheep anti-mouse igg is diluted to 0.8mg/mL with die buffer, by VacA antigen diluent
Liquid is sprayed on nitrocellulose filter 3 with stroke film gold spraying instrument with the amount of 1 μ L/cm, and the first detection line 6 is made in dry 0.5h；It will
CagA antigenic dilution is sprayed on nitrocellulose filter 3 with stroke film gold spraying instrument with the amount of 1 μ L/cm, and dry 0.5h is made second
Detection line 7；Urease antigenic dilution is sprayed on nitrocellulose filter 3 with stroke film gold spraying instrument with the amount of 1 μ L/cm, it is dry
Third detection line 8 is made in 0.5h；Sheep anti-mouse igg dilution is sprayed on nitrocellulose with stroke film gold spraying instrument with the amount of 1 μ L/cm
On film 3, nature controlling line 9 is made in dry 0.5h.
7, the assembling of detection reagent card
The sample pad 1 after the liner plate 5 of nitrocellulose membrane, step 1 optimization will be pasted with made from step 6, made from step 5
Quantum pad 2, blotting paper 4 assemble to obtain detection reagent card according to sequence from top to bottom, from inside to outside, as shown in Figure 1.
Comparative example 1
It is substantially same as Example 1, the difference is that: the quantum pad of embodiment 1 is replaced using gold-labelled pad and gold is marked
Pad the optimization without 1 step 2 of embodiment.
The minimum detection limit of the detection reagent card of comparative example 1 of the present invention: helicobacter Pylori urease Urease is resisted respectively
Sensitivity reference material 1:3,1:9,1:27 of body, cytotoxin-associated protein CagA antibody and vacuolating cytotoxin VacA antibody are dilute
It releases, the reference material detection of dilution 1:3,1:9 should be positive, and the reference material detection that dilution is 1:27 should be negative.
Table 1 is the minimum detection limit testing result of the detection reagent card of comparative example 1
As seen from Table 1, the sensitivity of the detection reagent card of comparative example 1 of the present invention is undesirable.
Comparative example 2
It is substantially same as Example 1, the difference is that: the polypropylene of embodiment 1 is replaced using dodecylamine hydrochloride
Amine hydrochlorate；The kayexalate of embodiment 1 is replaced using dodecyl sodium sulfate.
The negative match-rate of the detection reagent card of comparative example 2: take 15 parts of negative serums of clinical detection i.e. Urease antibody,
CagA antibody and all feminine genders of VacA antibody.
Table 2 is the negative match-rate testing result of the detection reagent card of comparative example 2
As seen from the table, the negative match-rate of the detection reagent card of comparative example 2 is 87%, there is the indeterminable risk of inspection.
Embodiment 2, classification of helicobacter pylori detection method
(1) test serum is added separately in the sample pad (1) of the detection reagent card of embodiment 1, reacts at room temperature 15min.
(2) it will test reagent card to be inserted into iSIA01 fluorescence immunity analyzer, read the yin and yang attribute of antibody.
Detection method and testing result
(1) it is gone to survey concentration value with the negative serum for meeting normal state of a large amount of three types respectively, due to being greater than 99%
Confidence level, therefore the mean value+3SD that every kind of serum is surveyed is set to positive cutoff value.
(2) minimum detection limit of the detection reagent card of the embodiment of the present invention 1: respectively by helicobacter Pylori urease Urease
Sensitivity reference material 1:3,1:9,1:27 of antibody, cytotoxin-associated protein CagA antibody and vacuolating cytotoxin VacA antibody
The reference material detection of dilution, dilution 1:3,1:9 should be positive, and the reference material detection that dilution is 1:27 should be negative.
Table 3 is the minimum detection limit testing result of the detection reagent card of embodiment 1
As seen from Table 3, the sensitivity of the detection reagent card of the embodiment of the present invention 1 meets the requirements.
(3) negative match-rate of the detection reagent card of embodiment 1: take 15 parts of i.e. Urease of negative serum of clinical detection anti-
Body, CagA antibody and all feminine genders of VacA antibody.
Table 4 is the negative match-rate testing result of the detection reagent card of embodiment 1
| Title | Urease antibody | CagA antibody | VacA antibody |
| Negative serum 1 | - | - | - |
| Negative serum 2 | - | - | - |
| Negative serum 3 | - | - | - |
| Negative serum 4 | - | - | - |
| Negative serum 5 | - | - | - |
| Negative serum 6 | - | - | - |
| Negative serum 7 | - | - | - |
| Negative serum 8 | - | - | - |
| Negative serum 9 | - | - | - |
| Negative serum 10 | - | - | - |
| Negative serum 11 | - | - | - |
| Negative serum 12 | - | - | - |
| Negative serum 13 | - | - | - |
| Negative serum 14 | - | - | - |
| Negative serum 15 | - | - | - |
As seen from Table 4, the negative match-rate of the detection reagent card of embodiment 1 is 100%.
(4) positive coincidence rate of the detection reagent card of embodiment 1: clinical detection positive serum i.e. Urease antibody positive is taken
Serum, CagA Positive Sera and each 13 parts of VacA Positive Sera.
Table 5 is the negative match-rate testing result of the detection reagent card of embodiment 1
As seen from Table 5, the positive coincidence rate of the detection reagent card of embodiment 1 is 100%
(5) detection reagent card and Western blot detection the clinical sample result of embodiment 1 compare
Table 6
As seen from Table 6, the testing result one of the testing result of the detection reagent card of embodiment 1 and the kit listed
It causes, and the detection reagent card of embodiment 1 is easy to operate in contrast, rapid reaction.
In above table ,+indicate positive ,-indicate negative.
The present invention is described in detail above, its object is to allow the personage for being familiar with this field technology that can understand this
The content of invention is simultaneously implemented, and it is not intended to limit the scope of the present invention, all Spirit Essence institutes according to the present invention
The equivalent change or modification of work, should be covered by the scope of protection of the present invention.
Claims (10)
1. a kind of classification of helicobacter pylori detection detection reagent card comprising sample pad (1), quantum pad (2), nitrocellulose
Film (3), blotting paper (4) and liner plate (5), it is characterised in that: the anti-of quantum dot microsphere label is coated on the quantum pad (2)
Former compound, the antigenic compound include Urease antigen, CagA antigen, VacA antigen and mouse IgG；The nitric acid is fine
It ties up on plain film (3) lateral the side where the blotting paper (4) from where the sample pad (1) and is disposed with the first detection
Line (6), the second detection line (7), third detection line (8) and nature controlling line (9) are coated with VacA in first detection line (6)
Antigen is coated with CagA antigen in second detection line (7), and it is anti-that Urease is coated in the third detection line (8)
Original, the nature controlling line are coated with sheep anti-mouse igg on (9).
2. classification of helicobacter pylori detection detection reagent card according to claim 1, it is characterised in that: the quantum
Point microballoon include quantum dot polystyrene microsphere, be deposited on the quantum dot Surfaces of Polystyrene Microparticles one or more layers is poly-
Electrolyte layer, the polyelectrolyte layer include the internal layer formed by polyallylamine hydrochloride and by kayexalate shape
At the outer layer for being deposited on the internal layer surface.
3. classification of helicobacter pylori detection detection reagent card according to claim 2, it is characterised in that: the quantum
Point microballoon is made by the steps to obtain:
(1) the quantum dot polystyrene microsphere is dispersed in water, polyallylamine hydrochloride solution, shaking table hatching 30 is added
~60min forms the internal layer of the polyelectrolyte layer, and centrifugation removes extra polyallylamine hydrochloride；
(2) step (1) the quantum dot polystyrene microsphere obtained for being formed with the internal layer is dispersed in water, polyphenyl is added
Vinyl sulfonic acid sodium solution, shaking table hatch the outer layer that 30~60min forms the polyelectrolyte layer, and centrifugation removes extra polyphenyl
Vinyl sulfonic acid sodium；
(3) step (1) and step (2) are selectively repeated, the quantum dot microsphere is obtained.
4. classification of helicobacter pylori detection detection reagent card according to claim 3, it is characterised in that: in step (1),
The mass ratio that feeds intake of the quantum dot polystyrene microsphere and the polyallylamine hydrochloride is 1:60~70；Step (2)
In, the formation has the quantum dot polystyrene microsphere of the internal layer and the quality that feeds intake of the kayexalate
Than for 1:60~70.
5. classification of helicobacter pylori detection detection reagent card according to any one of claim 1 to 4, feature exist
In: the quantum dot in the quantum dot microsphere is CdSe/CdS and/or CdSe/ZnS.
6. classification of helicobacter pylori detection detection reagent card according to claim 1, it is characterised in that: the antigen
The quality that feeds intake of Urease antigen described in compound, the CagA antigen, the VacA antigen and the mouse IgG
Than for 1:0.9~1.1:0.9~1.1:0.9~1.1；The quality that feeds intake of the quantum dot microsphere and the antigenic compound
Than for 1:0.6~1.
7. classification of helicobacter pylori detection detection reagent card according to claim 1, it is characterised in that: the quantum
Padding (2) is to pass through pretreated quantum pad, the pretreated method are as follows: it is slow that quantum pad (2) is immersed in the optimization of quantum pad
It is then dry and control humidity below 30% in fliud flushing, wherein the quantum pad optimization buffer include concentration be 45~
The trishydroxymethylaminomethane of 55mmol/L, the bovine serum albumin(BSA) that mass percentage concentration is 0.8~1.2%, quality percentage are dense
Degree for 0.4~0.6% polysorbas20, mass percentage concentration be 1.8~2.2% sucrose, mass percentage concentration be 0.08~
0.12% proclin300 and the purified water of surplus.
8. a kind of preparation side of the classification of helicobacter pylori detection detection reagent card as described in any one of claims 1 to 7
Method, characterized by the following steps:
(1) quantum dot microsphere is dispersed in phosphate buffer, is then added the antigenic compound, 10~40
DEG C shaking table hatches 1.5~2.5h, adds quantum dot microsphere confining liquid, hatches 2~3h, and centrifugal filtration obtains the quantum dot
The antigenic compound of microballoon label, wherein the quantum dot microsphere confining liquid includes the ox that mass percentage concentration is 8~12%
Seralbumin, the NaN that mass percentage concentration is 0.04~0.06%3And the purified water of surplus；
(2) the antigenic compound quantum dot dilution buffer of quantum dot microsphere label described made from step (1) is diluted
To 0.4~0.6 μm of ol/L, then it is sprayed on the amount of 0.8~1.2 μ L/cm on the quantum pad (2), it is dry that coating is made
The quantum pad (2) for the antigenic compound for thering is quantum dot microsphere to mark, wherein the quantum dot dilution buffer includes that concentration is
The trishydroxymethylaminomethane of 90~110mmol/L, the bovine serum albumin(BSA) that mass percentage concentration is 0.8~1.2%, quality hundred
Sucrose that polysorbas20 that point concentration is 0.4~0.6%, mass percentage concentration are 1.8~2.2%, mass percentage concentration 0.08
~0.12% proclin300 and the purified water of surplus；
(3) nitrocellulose filter (3) is pasted on the liner plate (5), by the VacA antigen, described
CagA antigen and the Urease antigen are diluted to 0.8~1.2mg/mL with die buffer respectively, by the sheep anti mouse
IgG is diluted to 0.6~1mg/mL with die buffer, is then sprayed on the nitric acid with the amount of 0.8~1.2 μ L/cm respectively
Cellulose membrane (3), dry obtained first detection line (6), second detection line (7), the third detection line
(8) and the nature controlling line (9), wherein the die buffer includes that concentration is 0.01~0.03mol/L, pH7~7.4
PBS buffer solution, mass percentage concentration be 0.8~1.2% trehalose, concentration be 8~12mmol/L ethylenediamine tetra-acetic acid,
The proclin300 that mass percentage concentration is 0.08~0.12%；
It (4) will treated is pasted with the liner plates (5) of nitrocellulose filter (3), sample pad (1), through step (2) through step (3)
Treated quantum pad (2), blotting paper (4) are assembled to obtain the detection reagent card.
9. preparation method according to claim 7, it is characterised in that:
The preparation method further includes carrying out pretreated step to the sample pad (1): sample pad (1) is immersed in sample
Product pad optimizes in buffer, then dries and controls humidity below 30%, wherein the sample pad optimizes buffer and includes
Polysorbas20 that trishydroxymethylaminomethane that concentration is 45~55mmol/L, mass percentage concentration are 0.4~0.6%, quality hundred
Divide the purified water of the concentration proclin300 for being 0.08~0.12% and surplus.
10. a kind of detection reagent as described in any one of claims 1 to 7 is stuck in answering in classification of helicobacter pylori detection
With.
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