CN201903546U - Colloidal gold immune detection test strip for detecting Francisella tularensis antibodies - Google Patents
Colloidal gold immune detection test strip for detecting Francisella tularensis antibodies Download PDFInfo
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- CN201903546U CN201903546U CN2010205466819U CN201020546681U CN201903546U CN 201903546 U CN201903546 U CN 201903546U CN 2010205466819 U CN2010205466819 U CN 2010205466819U CN 201020546681 U CN201020546681 U CN 201020546681U CN 201903546 U CN201903546 U CN 201903546U
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Abstract
The utility model discloses a colloidal gold immune detection test strip for detecting Francisella tularensis antibodies, which comprises a reaction support and a nitrocellulose membrane, wherein the nitrocellulose membrane is closely connected with the middle part of the upper surface of the reaction support. The upper surface of the start end of the nitrocellulose membrane is partly overlapped and connected with an absorbent pad, and the lower surface of the tail end of the nitrocellulose membrane is partly overlapped and connected with a golden-labeled antibody protective membrane. The lower surface of the golden-labeled antibody protective membrane is partly overlapped and connected with a sample pad, wherein the golden-labeled antibody protective membrane comprises a membrane body and a colloidal gold-labeled SPA coating uniformly coated on the membrane body. The nitrocellulose membrane is provided with a quality control strip enveloped by a goat anti rabbit IgG close to the start end and is provided with a detection strip enveloped by recombination FopA protein close to the tail end. The colloidal gold immune detection test strip for detecting the Francisella tularensis antibodies combines a colloidal gold-labeled and double-antibody sandwich immune chromatographic technique principle, adopts a dispersing agent and a sealing agent to pre-process the sample combination pad, has the advantages of simple operation, short detection time and the like, and is suitable for quick detection on site.
Description
Technical field
The utility model relates to a kind of colloid gold immune test strip that detects Francisella tularensis antibody.
Background technology
At present, when the double-antigen sandwiched colloidal golden method of domestic usefulness detection soil drew bacterium, soil commonly used drew the lipopolysaccharides (LPS) of bacterium as the antigen that detects antibody in the serum.But lipopolysaccharides has limited the promotion and application of these methods a large amount of obtaining, there is defective in aspects such as the pollution of environment and operator safety.And FopA albumen is a kind of high specificity and good outer membrane protein of conservative property that soil draws bacterium to have that have, and has good antigenicity, can be used as to detect the detection antigen that soil draws bacterium.
This experiment utilizes the recombinant antigen of preparation, and the colloidal gold immuno-chromatography test paper strip of employing indirect method detects soil and draws bacteria antibody, and specificity, the susceptibility of this method are estimated.By drawing the analog sample of bacterium FopA antibody to test, estimate this method and be used to detect the feasibility that soil draws bacterium in human serum, mixing soil.
The utility model content
The purpose of this utility model is to provide the colloid gold immune test strip of a kind of fast qualitative and detection by quantitative Francisella tularensis antibody.
For achieving the above object; the utility model detects the colloid gold immune test strip of Francisella tularensis antibody; comprise the reaction stilt; closely be connected in the nitrocellulose membrane of reaction stilt upper surface middle part; the upper surface of nitrocellulose membrane initiating terminal is overlapped with adsorptive pads and is connected; the lower surface of nitrocellulose membrane end is overlapped with golden labeling antibody diaphragm and is connected; the overlapping sample pad that is connected with of upper surface portion of gold labeling antibody diaphragm; wherein; gold labeling antibody diaphragm comprises membrane body; and be uniformly coated on the SPA coating of the colloid gold label on it, be provided with the Quality Control band of goat anti-rabbit igg bag quilt on the nitrocellulose membrane near its initiating terminal place; be provided with the test strip of reorganization FopA albumen bag quilt near its end.
Further, described reaction holder is the PVC plate, and adsorptive pads is a filter paper for oil.
Further, described golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
Further, described sample pad is glass fibre membrane, polyester film or filter paper fibre.
Further, the external packets of described test paper integral body is wrapped with shell, is provided with sample well corresponding to described sample pad place on this shell, is provided with view window corresponding to described Quality Control band, test strip place on the shell.
The utility model detects the colloid gold immune test strip of Francisella tularensis antibody, combine the know-why of colloid gold label and double antigens sandwich immunochromatography, adopt spreading agent and sealer to carry out pre-service the sample pad, determined optimum reaction condition, the susceptibility and the specificity of detection method have been guaranteed, have had simple to operately that detection time is short, need not the professional, be fit to advantages such as fast on-the-spot detection.And, by means of relevant device, can accomplish qualitative and detection by quantitative, thereby improve its promotion and application greatly.
Description of drawings
Fig. 1 is the range estimation sensitivity maps of the utility model test strip;
Fig. 2 is the specific detection Test Drawing;
Fig. 3 is the utility model test strip Detection of Stability figure;
Fig. 4 detects the fitting a straight line figure of test paper for the utility model;
Fig. 5 A is the front schematic view that the utility model detects test paper;
Fig. 5 B is the side structure synoptic diagram that the utility model detects test paper;
Fig. 6 A is the positive test symbol synoptic diagram that the utility model detects test paper;
Fig. 6 B is the negative result synoptic diagram that the utility model detects test paper;
Fig. 6 C is the invalid detection result schematic diagram that the utility model detects test paper.
Embodiment
As Fig. 1 to Fig. 4, shown in Fig. 5 A, Fig. 5 B, Fig. 6 A, Fig. 6 B, Fig. 6 C, Fig. 1 is the range estimation sensitivity maps of the utility model test strip.1: negative control; 2-7: soil draws bacteria antibody concentration 2400ng/mL, 1200ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL.
Fig. 2 is the specific detection test.The horse beautiful eyes GP2 antibody of test-strips 1-6:1ug/mL, smallpox M1R antibody, yellow hot YF-E-D3 antibody, bird flu NP antibody, dengue virus Deng-E-D3 antibody and Rickettsia prowazeki 120N antibody
Fig. 3 is the range estimation sensitivity maps Detection of Stability of the utility model test strip; Test-strips 1: negative control, 2～4: soil draws bacteria antibody concentration 120ng/mL, 60ng/mL, 30ng/mL
Among Fig. 5 A, Fig. 5 B, 1: adsorptive pads; 2: nitrocellulose membrane, T: bag is by reorganization FopA Protein Detection band; C: bag is by the Quality Control band of goat anti-rabbit igg; 3: the glass fibre membrane that contains colloid gold label SPA; 4: sample pad; 5: the reaction holder.
Fig. 6 A, Fig. 6 B, Fig. 6 C are testing result synoptic diagram of the present utility model; Two line positives of T, C; Line feminine gender of C; Invalid.
The utility model detects the colloid gold immune test strip of Francisella tularensis antibody, and the sensitivity of qualitative detection Francisella tularensis antibody reaches 30ng/ml.In analog sample detects, detect the sensitivity of adding variable concentrations Francisella tularensis antibody in the normal human serum and reach 30ng/ml.In addition, colloidal gold immuno-chromatography test paper strip of the present utility model is placed week back detection at 37 ℃ and is found that the detection sensitivity of test strips does not descend, and has shown good stable, is equivalent to 4 ℃ of effects of depositing 12 months; There is not the cross reaction specificity good to similar toxin such as horse beautiful eyes GP2 antibody, smallpox M1R antibody, yellow hot YF-E-D3 antibody, bird flu NP antibody, dengue virus Deng-E-D3 antibody and Rickettsia prowazeki 120N antibody.
Joining gold mark analyser has been set up the work matched curve that colloid gold immune layer standard measure detects Francisella tularensis antibody, in the range of linearity, i.e. and R
20.9905; The range of linearity 30～960ng/ml can carry out detection by quantitative.Find that after testing this method recovery height (41.0%～118.6%), repeatability are well.The collaurum quantitative detecting method that this research is set up can be finished the detection by quantitative in the certain limit in 15min, shortened 3～4 hours detection time.And false-positive phenomenon appears in the optimization of the whole detection system of process when having overcome the detection blood serum sample.
The utility model detects the colloid gold immune test strip of Francisella tularensis antibody, comprising with sample to be measured and sample diluting liquid mixing, sample mix liquid is added test paper sample well place, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result again; Described test strip comprises:
(1) reaction holder;
(2) adsorptive pads;
(3) nitrocellulose membrane, this film are coated with reorganization FopA albumen and Quality Control detection of antibodies band and Quality Control band;
(4) golden labeling antibody diaphragm wherein contains the SPA of colloid gold label;
(5) sample pad;
Wherein react holder and select the PVC plate for use; Adsorptive pads is selected filter paper for oil for use; Be provided with the Quality Control band of goat anti-rabbit igg bag quilt, be provided with the test strip of reorganization FopA albumen bag quilt near its end near its initiating terminal place on the nitrocellulose membrane.The material of gold labeling antibody diaphragm is selected from polyester film, glass fibre or filter paper fibre, contains the SPA of colloid gold label on it; The sample pad material is selected from polyester film, glass fibre or filter paper fibre.
Described adsorptive pads in the related test paper of the utility model method is selected filter paper for use, and described reaction holder is selected the PVC plate for use, and the material of described golden labeling antibody diaphragm is selected from polyester film, glass fibre or filter paper fibre, evenly is coated with the antibody of colloid gold label on it.
Described golden labeling antibody diaphragm in the related test paper of the utility model method is uniformly coated on polyester film, glass fibre membrane or the filter paper fibre film by the SPA with colloid gold label and makes.
The structure that the utility model detects the colloid gold immune test strip of Francisella tularensis antibody is: reaction holder 5 is positioned at bottom, nitrocellulose membrane 2 is positioned at the middle part on the reaction holder 5, the T place of nitrocellulose membrane 2 is test strips of reorganization FopA albumen bag quilt, and the C place is the Quality Control band of goat anti-rabbit igg bag quilt; Gold labeling antibody diaphragm 3 is positioned at a side on nitrocellulose membrane 2 tops and overlaps with it, and this film contains the reorganization Francisella tularensis antibody of colloid gold label; Adsorptive pads 1 is positioned at the opposite side for golden labeling antibody diaphragm 3 on nitrocellulose membrane 2 tops and overlaps with nitrocellulose membrane 2; Sample pad 4 is positioned on the nitrocellulose membrane 2 side opposite with adsorptive pads 1 and overlaps with golden labeling antibody diaphragm 3.
In the above-mentioned test paper, adsorptive pads 1 one sides are initiating terminal, and golden labeling antibody diaphragm 3 one sides are terminal, and test strip is positioned near terminal, and the Quality Control band approaches initiating terminal.
In the above-mentioned test paper, the concentration of reorganization FopA albumen is 1-5mg/ml, and the concentration of Quality Control goat anti-rabbit igg is 0.1-5mg/ml.
In the above-mentioned test paper, goat anti-rabbit igg antibody concentration is preferably 0.5-2.5mg/ml.
In the above-mentioned test paper, the amount of SPA mark 1ml collaurum is 5-6ug.
The utility model detects the preparation method of the colloid gold immune test strip of Francisella tularensis antibody, and this method comprises:
(1) uses the nitrocellulose membrane that sprays anti-Francisella tularensis antibody antibody and two bands of Quality Control antibody every stream metal spraying pen machine with certain spray film speed;
(2) a kind of golden labeling antibody diaphragm that contains the SPA of colloid gold label of preparation is uniformly coated on the SPA of colloid gold label on the glass fibre membrane, and after oven dry or the freeze drying, makes golden labeling antibody diaphragm.
Preparation method described in the utility model, in the step of wherein said spraying coated film, spray film speed is 10-100mm/s.
Preparation method described in the utility model, wherein said reorganization FopA albumen, its addition is 1-5mg/ml, the dilution of this albumen can be PB.
Preparation method described in the utility model, wherein the concentration of Quality Control goat anti-rabbit igg is 0.1-5mg/ml.
Preparation method described in the utility model, wherein the SPA with colloid gold label is uniformly coated on the glass fibre membrane, and wherein pH is 6.4-6.8.
The utility model relates to the application of described test paper in detecting Francisella tularensis antibody, comprising with sample to be measured and sample diluting liquid mixing, sample mix liquid is added test paper sample well place, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result again.
The utility model relates to the test paper of the detection Francisella tularensis antibody of method for preparing.
Use chromatography test-strips consumptive material in method described in the utility model and the product, consumptive material for example is the pad of glass fibre, nitrocellulose filter (for example NC film, SHF 1350225), and sample pad and adsorptive pads, filter paper can be available from Minipore companies.
The above-mentioned Francisella tularensis detection of antibodies test paper that the utility model relates to, it also comprises golden labeling antibody diaphragm.
The above-mentioned Francisella tularensis detection of antibodies test paper that the utility model relates to wherein reacts holder and selects the PVC plate for use.
The above-mentioned Francisella tularensis detection of antibodies test paper that the utility model relates to, wherein adsorptive pads is selected filter paper for use.
The above-mentioned Francisella tularensis detection of antibodies test paper that the utility model relates to, wherein golden labeling antibody diaphragm is selected polyester film, glass fibre or filter paper fibre for use.
One, the preparation of colloidal gold immuno-chromatography test paper strip
Material and method
1, antigen and antibody
The natural earth Lafranchise bacteria antibody of purifying, the rabbit of research department's preparation, mouse-anti reorganization jequirity A chain polyclonal antibody.
2, chromatography test-strips consumptive material
Pad (glass fibre), nitrocellulose filter (NC film, SHF 1350225), sample pad and adsorptive pads, filter paper are available from Minipore company.
3, experimental apparatus
Gold mark analyser (Shanghai Optics and Precision Mechanics institute, Chinese Academy of Sciences, China Inst. of Quarantine Inspection Sciences's joint research and development).
4, the preparation of colloidal gold immuno-chromatography test paper strip
4.1 collaurum pad
The SPA of pH value 6.4～6.8, concentration 0.2mg/ml is marked on the colloid gold particle that the sodium citrate legal system is equipped with the colloid gold particle of 25nm, 37 ℃ of dryings [7].
4.2 nitrocellulose filter
Detect band: reorganization FopA albumen 1mg/ml; Quality control band: goat anti-rabbit igg: 1mg/ml, 37 ℃ of dryings.
4.3 assembling
Sample pad, pad, adsorptive pads are attached to the end liner card that has the mixture that slits successively, are cut into the bar of 0.4cm, drying, dress shell, room temperature storage standby (referring to above-mentioned document).
Two, the qualitative and detection by quantitative of sample
1, the processing of analog sample
Adopt people's blood serum sample to detect, carry out after 100 times of dilutions as sample to be checked with sample preparation liquid; In addition, detect simultaneously adding FopA antibody in the human serum after the dilution.
2, qualitative detection
Sample and sample preparation liquid (as negative sample) 100ul after handling are added to the chromatography strip sample pad end for preparing, leave standstill 15min, observations.Detection band and quality control band redness all occurs and are judged to the positive, and it is negative that only redness appears in quality control band, and detection is with and quality control band does not all develop the color, and then are that test strips lost efficacy.
3, detection by quantitative
3.1 determining of decision content (CUT-OFF)
Press 1.6.1 negative sample is detected 20 times, read signal T/C ratio with the scanning of gold-marking immunity analyser.The mean value (AVERAGE) of 20 sample T/C ratios is the CUT-OFF value with 3 times of standard deviations (STDEVA) sum.
3.2 detection by quantitative is judged
Colloidal-gold strip after the colour developing is put into the scanning of gold-marking immunity analyser, read signal T/C ratio, positive greater than decision content.
Three, sensitivity test
1, detection by quantitative
1, the sensitivity of detection by quantitative
Detecting variable concentrations Francisella tularensis antibody, is 0.002 through computational discrimination value (CUT-OFF), and concentration is that average T/C ratio that the soil of 15ng/ml draws bacterium FopA to resist more is 0.001, is lower than 0.002, and is negative; How anti-the soil of 30～2400ng/ml draw bacterium FopA average T/C ratios to be respectively 0.03,0.061,0.122,0.211,0.367,0.627, and it is positive to be higher than 0.002 result; When soil drew the how anti-concentration of bacterium FopA greater than 30ng/ml, T/C average of relatives value was greater than 0.002, so detection sensitivity is 30ng/ml.
Each concentration testing result of table 1 Francisella tularensis antibody colloidal gold immunochromatographyassay assay test strips
2, the evaluation of visual inspection sensitivity
Prepare colloidal gold immuno-chromatography test paper strip by the optimum reaction condition of determining, detect the Francisella tularensis antibody of variable concentrations.Francisella tularensis antibody is diluted to concentration with sample preparation liquid is followed successively by concentration and is followed successively by 14.8 μ g/ml, 7.4 μ g/ml, 3.7 μ g/ml, 1.85 μ g/ml, 0.925 μ g/ml, 0.4625 μ g/ml, detect simultaneously.
The result as shown in Figure 1, directly the sensitivity 30ng/mL colour developing of range estimation is more clear.
3, analog sample detects
Adopt people's blood serum sample to detect, carry out after 100 times of dilutions as sample to be checked with sample preparation liquid; In addition, detect simultaneously adding FopA antibody in the human serum after the dilution.People's normal serum is negative, and it is 30ng/mL that interpolation soil draws the sample detection sensitivity of bacterium FopA antibody.
Four, typical curve match
Drawing the how anti-concentration of bacterium FopA with soil is horizontal ordinate (X), and the gold mark analyser value of reading T/C ratio (Y) is set up collaurum examination criteria curve for the ordinate mapping.When soil draws bacterium FopA antibody concentration to be 30～960ng/ml, be good linear relationship (Fig. 2) between the concentration of antibody and its gold mark analyser value of the reading T/C ratio, equation of linear regression is Y=0.0006X+0.0367, R2 is 0.9905.
Five, specificity test
Prepare colloidal gold immuno-chromatography test paper strip by the optimum reaction condition of determining, detect horse beautiful eyes GP2 antibody, smallpox M1R antibody, yellow hot YF-E-D3 antibody, bird flu NP antibody, dengue virus Deng-E-D3 antibody and Rickettsia prowazeki 120N antibody with the sample diluting liquid colloidal gold immuno-chromatography test paper strip that Processing of Preparation is good equally respectively, testing result sees 2.Positive reaction does not all appear in testing result, proves and the equal no cross reaction of these albumen that this detection method specificity is good.
Six, recovery test
In linear detection range, the soil that detects concentration known draws bacterium FopA antibody, calculates detectable concentration according to the gold mark analyser value of reading T/C ratio and typical curve, and the ratio percent of detectable concentration and theoretical concentration is the recovery.By table 2 as seen, etection theory concentration is 50,100,200,500, and when the soil of 800ng/ml drew bacterium FopA how anti-, its recovery was between 41.0%～118.6%.
The table 2 Francisella tularensis antibody colloidal gold immunochromatographyassay assay test strips detection system recovery
Seven, stability test
Referring to Fig. 3, freshly prepd colloidal gold immuno-chromatography test paper strip is deposited a week in 37 ℃, the soil with 120ng/mL, 60ng/mL, 30ng/mL concentration draws bacterium FopA antibody to detect respectively, and the result is shown in Fig. 5 .4.The soil of 120ng/mL, 60ng/mL, 30ng/mL draws bacterium FopA antibody to be positive reaction, and detection sensitivity is 30ng/mL.Detect with horse beautiful eyes GP2 antibody, smallpox M1R antibody, yellow hot YF-E-D3 antibody, bird flu NP antibody, dengue virus Deng-E-D3 antibody and Rickettsia prowazeki 120N antibody and to be negative reaction.This testing result is compared with freshly prepd test strip, and sensitivity does not obviously descend, and specificity is good.
Claims (5)
1. detect the colloid gold immune test strip of Francisella tularensis antibody; it is characterized in that; this test strips comprises the reaction stilt; closely be connected in the nitrocellulose membrane of reaction stilt upper surface middle part; the upper surface of nitrocellulose membrane initiating terminal is overlapped with adsorptive pads and is connected; the lower surface of nitrocellulose membrane end is overlapped with golden labeling antibody diaphragm and is connected; the overlapping sample pad that is connected with of bottom surface section of gold labeling antibody diaphragm; wherein; gold labeling antibody diaphragm comprises membrane body; and be uniformly coated on the SPA coating of the colloid gold label on it, be provided with the Quality Control band of goat anti-rabbit igg bag quilt on the nitrocellulose membrane near its initiating terminal place; be provided with the test strip of reorganization FopA albumen bag quilt near its end.
2. the colloid gold immune test strip of detection Francisella tularensis antibody as claimed in claim 1 is characterized in that described reaction holder is the PVC plate, and adsorptive pads is a filter paper for oil.
3. the colloid gold immune test strip of detection Francisella tularensis antibody as claimed in claim 1 is characterized in that described golden labeling antibody diaphragm is glass fibre membrane, polyester film or filter paper fibre.
4. the colloid gold immune test strip of detection Francisella tularensis antibody as claimed in claim 1 is characterized in that described sample pad is glass fibre membrane, polyester film or filter paper fibre.
5. the colloid gold immune test strip of detection Francisella tularensis antibody as claimed in claim 1, it is characterized in that, the external packets of described test paper integral body is wrapped with shell, be provided with sample well corresponding to described sample pad place on this shell, be provided with view window corresponding to described Quality Control band, test strip place on the shell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982776A (en) * | 2010-09-28 | 2011-03-02 | 中国检验检疫科学研究院 | Detection test paper for Francisella tularensis antibody, preparation method thereof, detection method utilizing detection test paper and quantitative detection system |
| CN111983229A (en) * | 2020-09-07 | 2020-11-24 | 三门县人民医院 | Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method |
-
2010
- 2010-09-28 CN CN2010205466819U patent/CN201903546U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982776A (en) * | 2010-09-28 | 2011-03-02 | 中国检验检疫科学研究院 | Detection test paper for Francisella tularensis antibody, preparation method thereof, detection method utilizing detection test paper and quantitative detection system |
| CN111983229A (en) * | 2020-09-07 | 2020-11-24 | 三门县人民医院 | Reagent strip for quantitatively detecting helicobacter pylori antibody by colloidal gold and detection method |
| WO2022048693A1 (en) * | 2020-09-07 | 2022-03-10 | 三门县人民医院 | Reagent strip for quantitatively detecting helicobacter pylori antibody by means of colloidal gold and detection method |
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