CN108061807A - It is a kind of to be used to measure immunofluorescent reagent item of CagA+ Hp antibody and its preparation method and application - Google Patents
It is a kind of to be used to measure immunofluorescent reagent item of CagA+ Hp antibody and its preparation method and application Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
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Abstract
The invention discloses a kind of for measuring immunofluorescent reagent item of CagA+ Hp antibody and its preparation method and application.The fluorescence of the present invention reagent strip is respectively there are three region：Sample application zone, land, detection zone.The reagent strip is respectively from top to bottom water absorption pad, chromatographic film, bonding pad, sample pad.It is characterized in that, film is sprayed on bonding pad a CagA antigens that fluorescent material CY3 is marked, C lines are coated with the monoclonal antibody of anti-CagA in chromatographic film, have high specific, available for CagA antibody in detection serum.Quantitative detection can be realized by being used together with fluorescence analyser, simple to operate.Available for test in laboratory, it can also be used to which field quick detection, detection only need about 10min.
Description
Technical field
The present invention relates to a kind of for measuring immunofluorescent reagent item and its preparation side of CagA+ Hp antibody
Method and application for detecting the concentration of the CagA+ Hp antibody in the patients serum of atherosclerosis, belong to and exempt from
Epidemiology detection field.
Technical background
Caused by atherosclerosis (Atherosclerosis, AS) is multifactor collective effect, pathogenesis is complicated,
It not yet illustrates completely at present.AS is a kind of chronic inflammation disease, and inflammatory mechanisms are present in the whole process of AS lesions, and inflammation is anti-
Should with immunological regulation during the entire process of AS occurrence and development important role.Platelet-activating factor acetylhydro-lase (Lp-PLA2),
Such as lysolecithin and oxidation free fatty, it is athero- to generate a variety of actuating arteries and veins for a variety of proinflammatory substances such as C reactive protein (CRP)
Induration, including endothelial cell apoptosis and Endothelial Dysfunction, the generation of stimulation adhesion factor and cell factor.
AS major risk crowds have Hypertensive Population, people with hyperlipidemia and a large amount of smoking populations, in addition also diabetes, fertilizer
Fat and inherent cause etc..The symptom of atherosclerosis depends primarily upon vascular lesion and the degree of ischemia of afflicted organ.
Helicobacter pylori (Helicobacter pylori, HP) infection is one of most common alimentary infection, Chinese HP
Infection rate is 34.52%~80.55%, average rate 58.07%.
Research shows that 60%~80% HP has the cytotoxic-associated gene A singly copied, generates cytotoxin
GAP-associated protein GAP A (cytotoxin associated gene A protein, CagA), CagA cause HP to have stronger cause a disease
Effect.
It is more and more to show that CagA positives HP infection be related with parenteral disease.HP infects and Atherosclerosis
Change forms related, and only CagA positive strains may be related with atherogenesis.It is athero- that actuating arteries and veins is infected for HP
The mechanism of correlation cardiovascular and cerebrovascular disease is hardened, correlative study proposes some may, it is believed that HP persistent infections result in inflammation
Factor activator and release make disorders of lipid metabolism that atherosclerotic plaque be promoted to be formed, and CagA directly acts on vascular wall and causes blood
Pipe proliferation of smooth muscle and local inflammation reaction cause endothelial injuries and dysfunction.
In consideration of it, establish it is a kind of effectively, it is quick, simple, sensitively detect CagA+ Hp (CagA) antibody
Method is of great significance.
The content of the invention
Present invention research is prepared for the antibody of high, high sensitivity the anticytotoxin GAP-associated protein GAP A of a species specificity, herein
On the basis of be prepared for it is a kind of for measuring the immunofluorescent reagent item of CagA+ Hp.
The object of the present invention is to provide it is a kind of for measure CagA+ Hp antibody immunofluorescent reagent item and
Its preparation method and application.For detecting the dense of the CagA+ Hp antibody in the patients serum of atherosclerosis
Degree.
To achieve the above object, present invention employs fluorescence immune chromatography principles to be prepared for fluorescence immune chromatography test paper bar,
The reagent strip is respectively there are three region：Sample application zone, land, detection zone.The structure of the reagent strip is by water absorption pad, chromatography
The order close adhesion of film, bonding pad, sample pad from top to bottom is on bottom plate.Antigenic compound (the Cy3- of fluorescent material mark
CagA) it is laid on bonding pad, being drawn in chromatographic film has detection line (T lines) and nature controlling line (C lines), and rabbit-anti people is coated on T lines
Anti- CagA monoclonal antibodies CA-1 is coated on IgG, C line.It drips and is flowed in the measuring samples liquid in sample pad under the action of capillary chromatography
To bonding pad, with the fluorescent antigen (Cy3-CagA) on bonding pad immune response occurs for the anti-CagA antibody of people in measuring samples
After form compound, then move to together on NC films, reach T lines when combined with the coated rabbit anti-human igg of T lines and be enriched with retention
At T lines, remaining unreacted fluorescent antigen is combined and is enriched with the coated anti-CagA monoclonal antibodies CA-1 of C lines and is retained at C lines,
Detection obtains testing result in immunofluorescence analysis instrument.Rabbit anti-human igg is the commercial antibodies of purchase.T lines detect patient
CagA antibody concentrations；At C lines, excessive Cy3-CagA antigenic compounds are specifically tied with mouse monoclonal antibody (i.e. anti-CagA monoclonal antibody CA-1)
It closes；The expression and production of CagA antigens are completed by Escherichia coli.
A kind of immunofluorescent reagent item for being used to measure CagA+ Hp antibody, it is characterised in that：Immunofluorescence
In reagent strip, water absorption pad, chromatographic film, bonding pad, sample pad are sequentially set；Sample pad corresponds to sample application zone, and bonding pad corresponds to
Land；Chromatographic film corresponds to detection zone；Chromatographic film is equipped with detection line T lines and nature controlling line C line；It is coated on detection line T lines
Rabbit anti-human igg；Anti- CagA monoclonal antibodies CA-1 is coated in nature controlling line C line；The antigen that tiling has fluorescent material to mark on bonding pad is answered
Close object Cy3-CagA.
T lines, C lines dilution formula of liquid are：0.01M PBS, 5% sucrose, 1%BSA；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；Cy3-CagA dilutes formula of liquid：0.01M
PBS, 0.5%PVA, 5% sucrose, 1%BSA；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1%Tween-20,
1%BSA.
Sample pad, bonding pad, chromatographic film, water absorption pad are a reagent strip unit, adjacent reagent cells overlap region
Length is 2mm, and sequentially on bottom plate, 3mm is cut into after completing bonding for close adhesion for water absorption pad, chromatographic film, bonding pad, sample pad
Wide reagent strip.
Water absorption pad is blotting paper；Chromatographic film is nitrocellulose (NC) film；Bonding pad and sample pad are glass fibre element
Film.
The distance between detection line T lines and nature controlling line C line are 10mm.Coated rabbit anti-human igg on detection line T lines, concentration
For 1mg/mL, dosage is 2 μ l/cm.Coated anti-CagA monoclonal antibodies CA-1 in nature controlling line C line, concentration 1mg/mL, dosage are 2 μ l/
cm。
The antigenic compound Cy3-CagA that the fluorescent material that concentration is 1mg/mL marks is made to tile (spray film) in bonding pad
On, the antigenic compound Cy3-CagA dosages of fluorescent material mark are 5 μ l/cm；The antigenic compound Cy3- of fluorescent material mark
In CagA, fluorescent material is cyanine dyes Cy3.
A kind of preparation method for the immunofluorescent reagent item for being used to measure CagA+ Hp antibody, including as follows
Step：
1) sample pad is handled：The sample pad of 20mm wide is soaked in sample pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Sample pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,1%BSA；
2) bonding pad is handled：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；
3) prepared by the antigenic compound Cy3-CagA of fluorescent material mark：By CagA antigens 0.15M NaCl solution room temperatures
Dialyse 4 it is small when, then with fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, by CagA antigens and 10mg/mL Cy3 according to
20:1 molar ratio room temperature is protected from light 45 minutes, is then protected from light dialysed overnight with 4 DEG C fresh of 0.15M NaCl solutions, then is used
0.01M PBS solution room temperatures be protected from light dialysis 4 it is small when, then be protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions, collect
Cy3-CagA is simultaneously preserved；
4) the antigenic compound Cy3-CagA spray films of fluorescent material mark：It is with Cy3-CagA dilutions that Cy3-CagA is dilute
It is interpreted as the Cy3-CagA solution that concentration is 1mg/mL and sprays film on bonding pad, the Cy3-CagA solution of 1mg/mL is on bonding pad
Dosage be 5 μ l/cm；Cy3-CagA dilutions：0.01M PBS, 0.5%PVA, 5% sucrose, 1%BSA；
5) T lines, the line of C lines：It takes and T lines is drawn on the NC films of 30mm wide, C lines, the distance of T lines and C lines is 10mm；It is wrapped on T lines
There is rabbit anti-human igg, be 1mg/mL with T lines, C lines dilution adjustment rabbit anti-human igg's concentration, dosage is 2 μ l/cm；It is wrapped on C lines
There is anti-CagA monoclonal antibodies CA-1, it is 1mg/mL to adjust anti-CagA monoclonal antibodies CA-1 concentration with T lines, C lines dilution, and dosage is 2 μ l/
cm；T lines, C line dilutions：0.01M PBS, 5% sucrose, 1%BSA；
6) assemble：By water absorption pad, chromatographic film, bonding pad, sample pad, sequentially close adhesion is on bottom plate, sample pad, combination
Pad, chromatographic film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is 2mm, after completing bonding
Cut into the reagent strip of 3mm wide.
The present invention also provides be used as detection people's blood for measuring the immunofluorescent reagent item of CagA+ Hp antibody
The application of the reagent of the concentration of CagA+ Hp antibody in clear.
Compared with the prior art, beneficial effects of the present invention are：Quantitative inspection can be realized by being used together with fluorescence analyser
It surveys, it is simple to operate.Available for test in laboratory, it can also be used to which field quick detection, detection only need about 10min；Using
The solution such as sample pad treatment fluid, bonding pad treatment fluid so that testing result is more stable and accurate.
Description of the drawings：
Fig. 1：A kind of structure diagram for the immunofluorescent reagent item for measuring CagA+ Hp；
Fig. 2：A kind of canonical plotting for the immunofluorescent reagent item for measuring CagA+ Hp；
Fig. 3：A kind of measure serum Ca g-A contents of immunofluorescent reagent item for measuring CagA+ Hp dissipate
Point diagram.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention will be described in detail.
A kind of immunofluorescent reagent item (Fig. 1) for being used to measure CagA+ Hp provided by the invention, reagent
Item and reagent include：
1. water absorption pad is blotting paper, water sorption is provided so that sample liquid is chromatographed to water absorption pad.
2. chromatographic film is nitrocellulose filter (NC films).It has higher affinity, drawn thereon detection line (T lines) and
Nature controlling line (C lines).
3. bonding pad is glass fibre element film.It can maintain the work of the antigenic compound (Cy3-CagA) of fluorescent material mark
Property, and cause in chromatography process so that Cy3-CagA is quickly redissolved and synchronously moved ahead with solution.
4. it is cyanine dyes Cy3 for marking the fluorescent material of anti-CagA antigens.
5. sample pad is glass fibre element film.It absorbs sample liquid, and the physical property for changing sample liquid maintains entire chromatography
The stabilization of system.
6. being coated with rabbit anti-human igg in detection line (T lines), concentration 1mg/mL, dosage is 2 μ l/cm.
7. being coated with anti-CagA monoclonal antibodies CA-1 on nature controlling line (C lines), concentration 1mg/mL, dosage is 2 μ l/cm.
The distance of 8.T lines and C lines is 10mm.
A kind of immunofluorescent reagent item for being used to measure CagA+ Hp provided by the invention, reagent strip system
Standby step is as follows：
1. sample pad is handled：The sample pad of 20mm wide is soaked in sample pad treatment fluid, when immersion 14 is small under the conditions of 4 DEG C
Overnight, taking-up be placed in 37 DEG C of thermostatic drying chambers dry 2 it is small when, preserve under the conditions of drying at room temperature.
2. sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1%Tween-20,1%BSA.
3. bonding pad is handled：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, when immersion 14 is small under the conditions of 4 DEG C
Overnight, taking-up be placed in 37 DEG C of thermostatic drying chambers dry 2 it is small when, preserve under the conditions of drying at room temperature.
4. bonding pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose,
1%BSA.
5.CY3 labelled antigens (Cy3-CagA)：By CagA antigens with 0.15M NaCl solutions room temperature dialysis 4 it is small when, then
With fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, by antibody and Cy3 (10mg/mL) according to 20:1 amount room temperature is protected from light instead
It answers 45 minutes, 4 DEG C then fresh of 0.15M NaCl solutions are protected from light dialysed overnight, are then protected from light with 0.01M PBS solution room temperatures
Dialyse 4 it is small when, be then protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions, collect Cy3-CagA and preserve.
6.CY3 labelled antigen sprays film：Cy3-CagA is diluted to fluorescent antigen dilution (i.e. Cy3-CagA dilutions)
Concentration is the Cy3-CagA solution of 1mg/mL and sprays film on bonding pad, and dosage is 5 μ l/cm.
7.CY3 labelled antigens dilution (i.e. Cy3-CagA dilutions)：0.01M PBS, 0.5%PVA, 5% sucrose, 1%
BSA。
8.T lines, the line of C lines：It takes and T lines is drawn on the NC films of 30mm wide, C lines, the distance of T lines and C lines is 10mm, is wrapped on T lines
There is rabbit anti-human igg, be 1mg/mL with dilution adjustment concentration, dosage is to be coated with anti-CagA monoclonal antibodies CA- on 2 μ l/cm, C lines
1, it is 1mg/mL with dilution adjustment concentration, dosage is 2 μ l/cm.
9.T lines, C line dilutions：0.01M PBS, 5% sucrose, 1%BSA.
10. assembling：By the order close adhesion of water absorption pad, chromatographic film, bonding pad, sample pad from top to bottom on bottom plate,
Each pad overlapping 2mm as shown in Figure 1,3mm wide reagent strips is cut into after completing bonding.
Embodiment one：The preparation of the specific monoclonal antibody of anti-CagA
1. animal immune：
The female Balb/c mouse of 6-8 week old are chosen as immunization, every mouse tail vein blood sampling before initial immunity, just
200 μ l of immunogene after intraperitoneal injection emulsification when secondary immune (antigen adds Freund's complete adjuvant, antigen CagA)；The 14th day after just exempting from,
It carries out in the same way second within 28th day and third time is immune (antigen adds Freund's incomplete adjuvant), the 5th day after the third immunization
Eye socket blood sampling is carried out to every mouse takes serum to assess antibody titer, carries out booster immunization, tail within the 7th day after the third immunization
Intravenous immune 100 μ l of original take splenic lymphocytes and SP2/0 (5 on the 3rd day after booster immunization:1 mixed proportion) carry out cell
Fusion, fusion is using PEG methods.After the completion of fusion based selective culture is cultivated with HAT.
2. subclone：
It is subcloned using limiting dilution assay.Feeder cells are laid in 96 orifice plates, by the hybridoma in positive hole,
Count, be diluted to 3 concentration with HT culture mediums, spread 96 orifice plates, each concentration 2 arranges so that in per hole cell be 1/hole, 3/
Hole, 10/hole, be placed in 37 DEG C, cultivated 7-10 days in 5%CO2 incubators, the positive most strong hole of selection is carried out continuously 2-3 times
Subclone, the anti-CagA monoclonal antibodies CA-1 of positive strain carry out turn hole and expand culture.
3. prepared by monoclonal antibody
Monoclonal antibody preparation employs in vivo method i.e. mice celiac inoculation, takes the ascites of mouse peritoneal, the antibody of acquisition is dense
Degree is high.The Balb/c female mices of 10 week old are taken, mouse peritoneal injects the 500 sterilized atoleines of μ l.Hybridoma passes through
Cross collection centrifugation, intraperitoneal injection 2 × 106A cell/mouse.Mouse web portion substantially swells within about 1 week or so, uses syringe needle
Ascites is adopted from abdominal cavity.
4. antibody purification
Employ the purifying that Protein G affinity columns carry out antibody.Ascites is diluted with isometric combination buffer,
Started column, and treated that sample flowed completely out, column is crossed with appropriate combination buffer, and treated that liquid is flowed completely out and cross column with appropriate eluent,
It simultaneously with the eluent in the small centrifuge tube of appropriate volume neutralizer and received is equipped with, is detected, collected blue with Coomassie brilliant blue
The deeper centrifuge tube antibody of color, loaded in bag filter, dialyses 3 days, and it (is used in nature controlling line C that albumen is detected after dialysis
The anti-CagA monoclonal antibody CA-1 of mouse) concentration, it is placed in -20 DEG C of preservations.
Embodiment two：A kind of standard curve for the immunofluorescent reagent item for measuring CagA+ Hp antibody
Configure a series of CagA+ Hp antibody titer of concentration gradients： 1RU/mL,2RU/mL,4RU/
ML, 8RU/mL, 16RU/mL, 32RU/mL, 64RU/mL take 50 μ l to be added drop-wise on immunofluorescent reagent item, then use detector
The fluorescence intensity of T lines and C lines is read, the fluorescence intensity ratio y of T/C and corresponding antibody concentration x are done into matched curve (figure respectively
2) fluorescence intensity formula corresponding with antibody concentration, is obtained, formula is：Y=0.1678x0.4335, related coefficient：R2=
0.981。
Embodiment three：A kind of clinical practice for the immunofluorescent reagent item for measuring CagA+ Hp
According to atherosclerosis index AI, (AI=[blood T-CHOL (TC)-high-density lipoprotein (HDL)] ÷ is highly dense
Lipoprotein (HDL) is spent, AI ＜ 4 reflect that the degree of artery sclerosis is not serious or mitigating, and the more arteriolosclerotic degree of numerical value is just
Lighter, the danger for triggering cardiovascular and cerebrovascular diseases is lower；The explanation of AI >=4 has occurred that artery sclerosis, and it is hard that numerical value gets over main artery
The degree of change is heavier, and the danger that cardiovascular and cerebrovascular diseases occur is higher.
The serum sample of 20 AI >=4, the serum sample of 30 AI ＜ 4, with exempting from for CagA+ Hp are selected
Epidemic disease fluorometric reagent item is measured, and is obtained measurement result and is drawn scatter diagram (Fig. 3).
The experimental results showed that the case for artery sclerosis has occurred, the concentration of the antibody of CagA+ Hp
It is worth higher in most cases.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of immunofluorescent reagent item for being used to measure CagA+ Hp antibody, it is characterised in that：Immunofluorescence is tried
In agent item, water absorption pad, chromatographic film, bonding pad, sample pad are sequentially set；Sample pad corresponds to sample application zone, and bonding pad corresponds to knot
Close area；Chromatographic film corresponds to detection zone；Chromatographic film is equipped with detection line T lines and nature controlling line C line；Rabbit is coated on detection line T lines
Anti-human igg；Anti- CagA monoclonal antibodies CA-1 is coated in nature controlling line C line；The antigen that tiling has fluorescent material to mark on bonding pad is compound
Object Cy3-CagA.
2. the immunofluorescent reagent item according to claim 1 for being used to measure CagA+ Hp antibody, feature
It is：T lines, C lines dilution formula of liquid are：0.01M PBS, 5% sucrose, 1%BSA；Bonding pad prescription for the treatment of liquid is：0.1M Tris-
HCl, 0.1% Tween-20,0.5%PVA, 5% sucrose, 1%BSA；Cy3-CagA dilutes formula of liquid：0.01M PBS, 0.5%
PVA, 5% sucrose, 1%BSA；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1% Tween-20,1%BSA.
3. the immunofluorescent reagent item according to claim 1 for being used to measure CagA+ Hp antibody, feature
It is：Sample pad, bonding pad, chromatographic film, water absorption pad are a reagent strip unit, adjacent reagent cells overlap zone length
For 2mm, sequentially close adhesion on bottom plate, cuts into 3mm's wide after completing bonding for water absorption pad, chromatographic film, bonding pad, sample pad
Reagent strip.
4. it is according to claim 1 a kind of for measuring the immunofluorescent reagent item of CagA+ Hp antibody,
It is characterized in that：Water absorption pad is blotting paper；Chromatographic film is nitrocellulose（NC）Film；Bonding pad and sample pad are glass fibre element
Film.
5. it is according to claim 1 a kind of for measuring the immunofluorescent reagent item of CagA+ Hp antibody,
It is characterized in that：The distance between detection line T lines and nature controlling line C line are 10mm.
6. it is according to claim 1 a kind of for measuring the immunofluorescent reagent item of CagA+ Hp antibody,
It is characterized in that：Coated rabbit anti-human igg on detection line T lines, concentration 1mg/mL, dosage are 2 μ l/cm.
7. it is according to claim 1 a kind of for measuring the immunofluorescent reagent item of CagA+ Hp antibody,
It is characterized in that：Coated anti-CagA monoclonal antibodies CA-1 in nature controlling line C line, concentration 1mg/mL, dosage are 2 μ l/cm.
8. it is according to claim 1 a kind of for measuring the immunofluorescent reagent item of CagA+ Hp antibody,
It is characterized in that：The antigenic compound Cy3-CagA that the fluorescent material that concentration is 1mg/mL marks is made to spray film on bonding pad, fluorescence
The antigenic compound Cy3-CagA dosages of mass signatures are 5 μ l/cm；In the antigenic compound Cy3-CagA of fluorescent material mark,
Fluorescent material is cyanine dyes Cy3.
9. a kind of immunofluorescence examination for being used to measure CagA+ Hp antibody described in claim 1-8 any one
The preparation method of agent item, includes the following steps：
Sample pad processing：The sample pad of 20mm wide is soaked in sample pad treatment fluid, soaked overnight under the conditions of 4 DEG C, after taking-up
Drying in 37 DEG C of thermostatic drying chambers is placed in, is preserved under the conditions of drying at room temperature；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl,
0.1% Tween-20,1%BSA；
Bonding pad processing：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, soaked overnight under the conditions of 4 DEG C, after taking-up
Drying in 37 DEG C of thermostatic drying chambers is placed in, is preserved under the conditions of drying at room temperature；Bonding pad prescription for the treatment of liquid is：0.1M Tris-HCl,
0.1% Tween-20,0.5%PVA, 5% sucrose, 1%BSA；
It is prepared by the antigenic compound Cy3-CagA of fluorescent material mark：By the 0.15M NaCl solutions room temperature dialysis 4 of CagA antigens
Hour, then with fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, by CagA antigens and 10mg/mL Cy3 according to 20:1
Molar ratio room temperature is protected from light 45 minutes, is then protected from light dialysed overnight with 4 DEG C fresh of 0.15M NaCl solutions, then is used 0.01M
PBS solution room temperature be protected from light dialysis 4 it is small when, then be protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions, collect Cy3-CagA
And it preserves；
The antigenic compound Cy3-CagA spray films of fluorescent material mark：Cy3-CagA is diluted to concentration with Cy3-CagA dilutions
For 1mg/mL Cy3-CagA solution and spray film on bonding pad, the dosage of the Cy3-CagA solution of 1mg/mL on bonding pad is
5μl/cm；Cy3-CagA dilutions：0.01M PBS, 0.5%PVA, 5% sucrose, 1%BSA；
T lines, the line of C lines：It takes and T lines is drawn on the NC films of 30mm wide, C lines, the distance of T lines and C lines is 10mm；Rabbit is coated on T lines
Anti-human igg, is 1mg/mL with T lines, C lines dilution adjustment rabbit anti-human igg's concentration, and dosage is 2 μ l/cm；It is coated on C lines anti-
CagA monoclonal antibody CA-1, it is 1mg/mL to adjust anti-CagA monoclonal antibodies CA-1 concentration with T lines, C lines dilution, and dosage is 2 μ l/cm；T lines, C
Line dilution：0.01M PBS, 5% sucrose, 1%BSA；
Assembling：By water absorption pad, chromatographic film, bonding pad, sample pad, sequentially close adhesion is on bottom plate, sample pad, bonding pad, chromatography
Film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is 2mm, is cut into after completing bonding
The reagent strip of 3mm wide.
10. the immunofluorescent reagent item for being used to measure CagA+ Hp antibody described in claim 1-8 any one
The application of the reagent of concentration as the CagA+ Hp antibody in detection human serum.
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| CN113655220A (en) * | 2021-08-19 | 2021-11-16 | 浙江嘉孚生物科技有限公司 | Time-resolved fluorescence immunochromatographic test paper for detecting invasive aspergillus infection and preparation method thereof |
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Application publication date: 20180522 |