CN101666799A - Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography - Google Patents
Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography Download PDFInfo
- Publication number
- CN101666799A CN101666799A CN200910181759A CN200910181759A CN101666799A CN 101666799 A CN101666799 A CN 101666799A CN 200910181759 A CN200910181759 A CN 200910181759A CN 200910181759 A CN200910181759 A CN 200910181759A CN 101666799 A CN101666799 A CN 101666799A
- Authority
- CN
- China
- Prior art keywords
- line
- antibody
- film
- escherichia coli
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography, belonging to the technical field of antibody detection kits and consisting of colloidal gold cushion prepared by utilizing a colloidal gold solution the granule of which is 20nm to mark purified sheep anti-mouse IgG, nitric acid cellulose films which are respectively coated by a colibacillus O157:H7 antigen and purified mouse serum IgG and serve as a detection line and a quality control line, a detection test strip formed by assembling a sample absorbing cushionprepared from glass cellulose films and an absorbing cushion made of water absorbing filter paper, positive serum, negative serum, a serum sample diluent and a test strip operating instruction. The test strip is convenient to use, rapid and sensitive, and a detection result can be obtained within 10 minutes, and therefore, the test strip is suitable for on-site detection when infection status ofmouse O157:H7 is surveyed and traced back in animal farmer areas, food production enterprise areas and the like and use when the food safety environment risk evaluation is carried out.
Description
One, technical field
The present invention relates to a kind of Escherichia coli O 157: H7 mouse serum antibody colloidal gold immunochromatographydetection detection test paper bar belongs to antibody assay kit technology territory.Be exclusively used in Escherichia coli O 157 in the mouse serum: the fast detecting of H7 antibody, the scene when being applicable to community surveys such as animal-breeding field, food production enterprise and reviewing mouse O157:H7 infection state is detected and is used when the risk assessment of conducting food security context.
Two, background technology
Escherichia coli (Escherichia coli, E.coli) O157:H7 is a kind of important Amphixenosis's pathogenic microorganism, the people infects O157:H7 can suffer from diarrhoea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombus decrease of platelet purpura severe complications such as (TTP), severe patient can cause death (Xia Xingxia, Liu Jie, Wang Yongshan, Zhu Yumei, Ou Yangwei, what Kong Wang, Zhang Xuehan. animal derived people beast suffers from the research of the biological novel formulation of bacterial disease prevention and control altogether: the foundation of the I. secretion enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College monoclonal antibody hybridoma cell strain. Jiangsu agricultural journal, 2009,25 (2): 291-295).Nineteen eighty-two finds first in the U.S., in after this more than 20 year, all distributed in worldwide and broken out by the disease due to the O157:H7, and 1996 years once in Japanese outbreak of epidemic.Calendar year 2001, on Chinese Jiangsu, Anhui and other places the source sexuality having taken place also repeatedly to eat has dyed the O157:H7 incident, cause 177 people's death, caused the extensive concern of the whole society.The infection of O157:H7 is because of having outbreak of epidemic trend, strong pathogenic and lethal and antibiotic therapy characteristics such as may aggravate one's illness, become global public health problem, the World Health Organization (WHO) has classified O157:H7 as new foodborne bacterial pathogens.
Food source property and to contact with animal carrier be the main path that the people infects O157:H7, ruminant is the main host of O157:H7, positive rate 0.1%～16%, in addition, also separable to O157:H7 from the ight soil of goose, chicken, horse, deer, sea-gull, sheep, goat, pigeon, duck, rabbit, dog, cat.Mouse and food production and human lives are in close relations, play an important role in eqpidemic disease is propagated, and food security and human health are threatened greatly, infect the situation of O157:H7 and the research data of epidemiological significance thereof but lack relevant mouse so far.
The present invention is based on the severe situation of China's food security and to the active demand of quick detection kit, utilization colloidal gold immunochromatographimethod technology, preparation mouse serum O157:H7 antibody colloidal gold immunochromatographyassay assay test strip, for the infection state of regional mouse O157:H7 such as monitoring animal-breeding field, food production enterprise provides simple and rapid antibody detection method, O157:H7 epidemiology is reviewed in order to carry out, investigation and food security environmental risk assessment provide technical support.
Three, summary of the invention
Technical matters
The present invention is directed to the more environment present situations of regional mouse such as current China part animal-breeding field, food production enterprise, develop a kind of easy, quick and be suitable for the on-the-spot monitoring mouse Escherichia coli O 157 that uses: H7 antibody colloidal gold immunochromatographyassay assay test strip is used for the O157:H7 epidemiology survey, reviews and the food security environmental risk assessment.
Technical scheme specific embodiments of the present invention is as follows:
A kind of Escherichia coli (Escherichia coli, E.coli) O157:H7 mouse serum antibody colloidal gold immunochromatographydetection detection test paper bar is that the sheep anti-mouse igg of the colloidal gold solution mark purifying of 20nm is made the collaurum pad, formed as test strip, positive serum, negative serum, serum samples diluted liquid and test strips operation instructions that detection line and nature controlling line, absorption of sample pad that the plain film of glass fibre is made, absorption pad that absorbent filter is made are assembled into the nitrocellulose filter of the mouse serum IgG bag quilt of O157:H7 antigen and purifying respectively by particle.
Above-mentioned test strip, its preparation method is:
1) Escherichia coli O 157: the preparation of H7 antigen
Cultivate Escherichia coli O 157: H7, the centrifugal 30min of 3500r/min, resuspended with PBS, 0.5% formalin-inactivated, PBS are washed 3 times, and precipitation is resuspended in PBS, and-20 ℃ are frozen standby;
2) preparation of immune colloid gold
(absorbance value when wavelength 521nm is 1.847, A to get 20nm particle colloidal gold solution
521=1.847) 20ml, under magnetic agitation (200r/min), slowly add the sheep anti-mouse igg of purifying, at room temperature stir 30min, adding mass volume ratio 10% bovine serum albumin(BSA) BSA, to make its final concentration be mass volume ratio 0.4%, stirring at room 5min, adding mass volume ratio 10%PEG, to make the PEG final concentration be mass volume ratio 0.2%, stirring at room 5min; The centrifugal 45min of 12000r/min draws centrifugal supernatant, and precipitation is dissolved in 2ml preserves in the liquid, and with 0.45 μ m membrane filtration, it is standby to put 4 ℃ of preservations, makes colloid gold label antibody stoste;
3) preparation of the plain film of golden labeling antibody glass fibre
Get colloid gold label antibody stoste 1ml, add working fluid 2ml dilution, mixing is added on the plain film of glass fibre equably, makes the plain film of golden labeling antibody glass fibre, i.e. collaurum pad, and 37 ℃ of dryings, 4 ℃ of preservations are standby;
4) preparation of solid phase nitrocellulose filter NC
PBS with 0.01M pH7.2 dilutes Escherichia coli O 157 respectively: the mouse serum IgG of H7 antigen and purifying, with protein concentration is the 2.0mg/ml Escherichia coli O 157: the mouse serum IgG point sample of H7 antigen and purifying on the NC film respectively as detection line and nature controlling line, drying at room temperature, sealing, the chromatography test background is clear;
5) assembling of test strips
On wide by 2 * long 9cm PVC base plate (1), interlude is pasted wide by 2 * long 3cm solid phase nitrocellulose filter, i.e. NC film (2); Paste collaurum-sheep anti-mouse igg antibody bond glass fibre membrane (5) of 2 * 2cm near the PVC backplate surface of solid phase cellulose membrane detection line T line one end, plain film of this glass fibre and the overlapping 0.1cm of NC film (2), the overlapping 0.2cm of absorption of sample pad (7) of the other end and 2 * 1cm; The PVC backplate surface of close solid phase cellulose membrane nature controlling line C line one end is pasted the thieving paper (6) of 2 * 3.5cm, with the overlapping 0.1cm of NC film; Antibody solid phase nitrocellulose filter NC film (2) is gone up to draw an Escherichia coli O 157: a H7 Detection of antigen line T line (3) and a mouse serum IgG nature controlling line C line (4).
The T line is drawn on the position of long 3cm * wide 2cm NC film one side 1cm, with concentration is the Escherichia coli O 157 of 2.0mg/mL: H7 antigen is packed into and is drawn the film machine, be sprayed on the NC film, another shower nozzle is drawn nature controlling line C line, concentration is the mouse serum IgG of 2.0mg/mL, the C line is drawn on the position of NC film opposite side 1cm, the distance of detection line and nature controlling line is 1cm, the point sample amount is 5 μ l and is sprayed on the nitrocellulose membrane, the cardboard that assembles is by longitudinal shear, be cut into the strip that width is 2.5mm, be antibody test test strips finished product.
The usage of above-mentioned test strip is characterized in that:
When detecting antibody, mouse serum sample to be checked is diluted 100 times with the sample diluting liquid that contains multi-joint bacterial immunity rabbit anteserum, get 200 μ l and drip reaction zone in test strips, if red stripes all occurs at detection line place and nature controlling line place, show and contain Escherichia coli O 157 in the serum sample to be checked: H7 antibody is judged to the positive; If only red stripes occurs at the nature controlling line place, show not contain Escherichia coli O 157 in the sample to be checked: H7 antibody then is judged to feminine gender; If red stripes does not appear in nature controlling line,, represent that all the failure of an experiment or test strips lost efficacy no matter whether detection line red stripes occurs.
In the usage of above-mentioned test strip, use the PBS solution dilution of the 0.01M pH7.2 that contains volume ratio 2% multi-joint bacterial immunity rabbit anteserum to be tried the mouse serum sample.
Beneficial effect characteristics of the present invention and advantage are as follows:
Escherichia coli O 157: H7 is a kind of pathogen of propagating and infecting through the food source, sets up fast, responsive, special detectable is one of important measures that prevention and control should disease.At present, the detectable of development all to detect antigen or nucleic acid, still lacks antibody detection method both at home and abroad.
The present invention is directed to the subject matter of current China food sanitation safe and to the active demand of quick detection kit, the colloidal gold immunochromatographimethod technology of uses advanced, set up Escherichia coli O 157: H7 mouse serum antibody colloidal gold immunochromatographydetection detection test paper bar, this test strips is easy to use, quick, special, responsive, practical, can obtain the result in 10 minutes, the O157:H7 epidemiology survey, review and the food security environmental risk assessment aspect have important use and be worth.
Four, description of drawings
The structure of Fig. 1 colloidal gold immuno-chromatography test paper strip
1.PVC base plate; 2. nitrocellulose filter (NC film); 3. detection line; 4. nature controlling line; 5. collaurum pad; 6. absorption pad (absorbent filter); 7. absorption of sample pad (glass fibre plain film)
The criterion as a result of Fig. 2 colloidal gold immuno-chromatography test paper strip
Fig. 3. the testing result of test strips
1. water; 2. negative mouse serum; 3. positive mouse serum
Fig. 4. the sensitivity Detection result of test strips
1. negative control; 2-7. being respectively positive mouse serum dilution is 1: 1 * 10
7～1: 1 * 10
2
Five, embodiment
1 experiment material
(absorbance value when wavelength 521nm is 1.847, A for the plain film of glass fibre, cellulose nitrate (NC) film and 20nm particle colloidal gold solution
521=1.847) available from the prompt peaceful bio tech ltd in Shanghai;
The mouse serum IgG of purifying system with the sulfuric acid acid ammonium salt analyse with DEAE cellulose method (Liu Yubin, Gou Shijin edits. the animal immunology experimental technique. Changchun: Jilin science tech publishing house, 1989,8-15) from mouse serum, extract;
Behind the mouse serum IgG injecting immune goat of purifying sheep anti-mouse igg system usefulness purifying 4 times, the preparation immune serum is further analysed and DEAE cellulose method purifying with the sulfuric acid acid ammonium salt;
Multi-joint bacterial immunity rabbit anteserum system is with Bacterium enteritidis (Salmonella enterica), Escherichia coli (Escherichia coli) K88, K99, O159, O141, TOP10, B121, BJ5183 and Streptococcus suis (Streptococcus suis) SS-1 hybrid antigen (described bacterial strain is all available from Nanjing Tianbang Bio-industry Co., Ltd.), the rabbit anteserum behind the injecting immune 4 times;
50 parts of O157:H7 immune mouse serums (positive serum) are with the preparation of O157:H7 immunity BALB/c mouse method; 20 parts of negative serums pick up from cleaning level mouse.Above animal is all available from Yangzhou University comparative medicine center.
2 Escherichia coli O 157s: the preparation of H7 antigen
A large amount of Escherichia coli O 157: H7500mL that cultivate, the centrifugal 30min of 3500r/min, resuspended with PBS, 0.5% formalin-inactivated, PBS are washed 3 times, and precipitation is resuspended in PBS,-20 ℃ of frozen standby (Xia Xingxia, Liu Jie, Wang Yongshan, Zhu Yumei, Ou Yangwei, what Kong Wang, Zhang Xuehan. animal derived people beast suffers from the research of the biological novel formulation of bacterial disease prevention and control altogether: the foundation of the I. secretion enterobacteria O157:H7 of Chinese People's Anti-Japanese Military and Political College monoclonal antibody hybridoma cell strain. Jiangsu agricultural journal, 2009,25 (2): 291-295).
The preparation of 3 immune colloid golds
3.1 the best pH of colloid gold label measures
Get 1mg/mL sheep anti mouse 3 μ L, add 100 μ L pH values respectively and be in 7.0,8.0,9.0,10.0 the collaurum, mixing is placed 10～15min under the room temperature; Add 10%NaCl solution 20 μ L more respectively, mixing leaves standstill observations behind the 2h, determines golden mark optimum pH.When collaurum pH value was 7.0,8.0,9.0,10.0, immune colloid gold is solution-stabilized, and was bright-colored, and the pH value is 7.0 (the mark optimal pHs of using in the step 3.3) during colloid gold label albumen.
3.2 the mensuration of the suitableeest labelled protein concentration
Get 10 test tubes, each adds the 1.0ml colloidal gold solution.Sheep anti-mouse igg is diluted to 50 μ g/ml, 45 μ g/ml, 40 μ g/ml, 35 μ g/ml, 30 μ g/ml, 25 μ g/ml, 20 μ g/ml, 15 μ g/ml, 10 μ g/ml respectively with 0.01M PB pH7.2, respectively gets 1.0ml and be sequentially added in the above-mentioned colloidal gold solution.Control tube adds 1ml dilution (not containing protein), mixing.After placing 5min, in above-mentioned every test tube, add 10% sodium-chloride water solution 0.1ml.The mixing room temperature left standstill 1～2 hour, observations.The minimum albumen consumption of stable colloid gold is 25 μ g/ml.Increase by 10%～20% actual amount that is albumen to be marked on this basis again, the used protein content of this test is 27.5 μ g/ml (the suitableeest protein concentrations of mark that step 3.3 is used).
3.3 the mark of collaurum
Get 20nm particle colloidal gold solution 20ml, under magnetic agitation, slowly add the sheep anti-mouse igg of purifying, at room temperature stir 30min.Adding 10%BSA, to make its final concentration be 0.4%, stirring at room 5min.Adding 10%PEG, to make its final concentration be 0.2%, stirring at room 5min.The centrifugal 45min of 12000r/min carefully draws centrifugal supernatant, and precipitation is dissolved in 2ml and preserves (preservation liquid making method: 0.1g sodium tetraborate, 0.25g BSA, 0.025g NaN in the liquid
3PH to 7.4 is transferred with 6N HCl in the back that is dissolved in water, and moisturizing is to 250ml, behind 0.45 μ m membrane filtration, 4 ℃ of preservations), with 0.45 μ m membrane filtration, it is standby to put 4 ℃ of preservations.
4 Escherichia coli O 157s: the preparation of H7 antibody colloidal gold immunochromatographyassay assay test strips
4.1 the preparation of the plain film of golden labeling antibody glass fibre
Get golden labeling antibody stoste 1ml, add working fluid 2ml dilution (working fluid compound method: 6.1gNa
2HPO
4.12H
2After being dissolved in water, O, 8.5g NaCl, 5.0g PVP40,2.1g boric acid, 1.0g PEG4000,0.5g BSA transfer pH to 7.4 with 6N HCl, moisturizing is to 1000ml, behind 0.45 μ m membrane filtration, 4 ℃ of preservations), mixing is added on the plain film of glass fibre uniformly, makes the plain film (collaurum pad) of golden labeling antibody glass fibre, 37 ℃ of dryings, 4 ℃ of preservations are standby.
4.2 the preparation of solid phase nitrocellulose filter (NC)
PBS with 0.01M pH7.2 dilutes Escherichia coli O 157 respectively: the mouse serum IgG of H7 antigen and purifying, with different protein concentration point samples on the NC film respectively as detection line and nature controlling line, carry out mark, drying at room temperature at an end of detection line.With the O157:H7 immune mouse serum is that test sample is carried out chromatography test, observes the colour developing situation, optimizes the point sample protein concentration.With protein concentration is the 2.0mg/ml Escherichia coli O 157: respectively as detection line and nature controlling line, seal by drying at room temperature on the NC film for the mouse serum IgG point sample of H7 antigen and purifying, and the chromatography test background is clear.
4.3 the assembling of test strips
As shown in Figure 1, on wide by 2 approximately * long 9cm PVC base plate (1), interlude is pasted wide by 2 * long 3cm solid phase nitrocellulose filter (NC film 2); Paste collaurum-sheep anti-mouse igg antibody bond glass fibre membrane (5) of 2 * 2cm near the PVC backplate surface of solid phase cellulose membrane detection line (T line) end, plain film of this glass fibre and the overlapping 0.1cm of NC film (2), the overlapping 0.2cm of absorption of sample pad (7) of the other end and 2 * 1em; Close solid phase cellulose membrane nature controlling line (C line) the PVC backplate surface of an end is pasted the thieving paper (6) of 2 * 3.5cm, with the overlapping 0.1cm of NC film; Antibody solid phase nitrocellulose filter (NC film 2) is gone up to draw an Escherichia coli O 157: a H7 Detection of antigen line T line (3) and a mouse serum IgG nature controlling line C line (4).
The T line is drawn on the position of long 3cm * wide 2cm NC film one side 1cm, with concentration is the Escherichia coli O 157 of 2.0mg/mL: H7 antigen is packed into and is drawn the film machine, be sprayed on the NC film, another shower nozzle is drawn nature controlling line C line, concentration is the mouse serum IgG of 2.0mg/mL, the C line is drawn on the position of NC film opposite side 1cm, and the distance of detection line and nature controlling line is 1cm, and the point sample amount is 5 μ l and is sprayed on the nitrocellulose membrane.The cardboard that assembles is cut into the strip that width is 2.5mm by longitudinal shear, is antibody test test strips finished product.
4.4 the use of test strips
When antibody test, mouse serum sample to be checked is carried out dilution in 1: 100 with the 0.01MPBS pH7.2 solution that contains 2% multi-joint bacterial immunity rabbit anteserum, get 200 μ l and drip reaction zone in test strips, if red stripes all occurs at detection line place and nature controlling line place, show and contain Escherichia coli O 157 in the sample to be checked: H7 antibody is judged to the positive; If only red stripes occurs at the nature controlling line place, show not contain Escherichia coli O 157 in the sample to be checked: H7 antibody then is judged to feminine gender; If red stripes does not appear in nature controlling line,, all represent the failure of an experiment or test strips inefficacy (Fig. 2) no matter whether detection line red stripes occurs.Detect the positive mouse serum of O157:H7, negative mouse serum and three kinds of test sample of water respectively with the test strips that is assembled into, have only the positive mouse blood serum sample of O157:H7 red stripes to occur at the detection line place, the result is clear easily to distinguish (Fig. 3).
The specific detection of 5 colloidal gold immuno-chromatography test paper strips
Detect Escherichia coli O 157 with test strips: H7 immune mouse serum sample, all positive; Detect mouse serum, cleaning level mouse serum and the pure water of salmonella, Escherichia coli K88, K99, O159, O141, TOP10, BL21, BJ5183 and streptococcus SS-1 immunity, all negative, show that this test strips has good specificity.
The sensitivity Detection of 6 colloidal gold immuno-chromatography test paper strips
With Escherichia coli O 157: H7 immune mouse serum sample is from 1: 1 * 10
2Logarithm doubling dilution to 1: 1 * 10
7, each dilutability is got 200 μ l difference point sample in the point sample place of colloid gold immune test paper bar, observations behind the 10min.Use the antigen coated indirect ELISA of Escherichia coli O 157: H7 to detect simultaneously, relatively both testing results.Detect 1: 1 * 10 with test strips
2～1: 1 * 10
6Dilution O157:H7 immune mouse serum red stripes all occurs at detection line and nature controlling line place, shows that the antibody test result is positive; And 1: 1 * 10
7Dilution serum only tangible red stripes occurs at the nature controlling line place, shows that the antibody test result is negative, identical with negative control (Fig. 4).In ELISA parallel control test experience, tiring of positive mouse serum is 1 * 10
5, suitable with the susceptibility of colloidal gold immuno-chromatography test paper strip detection method.This test strips is easy and simple to handle, quick, can obtain the result in 10 minutes; And detect with ELISA, need 2 hours from adding serum to be checked at least to result of determination.
The Detection of Stability of 7 colloidal gold immuno-chromatography test paper strips
Test strips is placed in the exsiccator, and 4 ℃ of hermetically dryings are preserved, and are a collection of every the 30d extraction, the positive and negative mouse serum sample of parallel detection with freshly prepd test strips, relatively both testing results.After 6 months, with the freshly prepd test strips comparison and detection positive and negative mouse serum, all find to reduce, and illustrates that this test strips has good stable by specificity and susceptibility 4 ℃ of kept dry for colloidal gold strip.
Claims (4)
1, a kind of Escherichia coli (Escherichia coli, E.coli) O157:H7 mouse serum antibody colloidal gold immunochromatographydetection detection test paper bar is that the sheep anti-mouse igg of the colloidal gold solution mark purifying of 20nm is made the collaurum pad, formed as test strip, positive serum, negative serum, serum samples diluted liquid and test strips operation instructions that detection line and nature controlling line, absorption of sample pad that the plain film of glass fibre is made, absorption pad that absorbent filter is made are assembled into the nitrocellulose filter of the mouse serum IgG bag quilt of O157:H7 antigen and purifying respectively by particle.
2, test strip according to claim 1, its preparation method is:
1) Escherichia coli O 157: the preparation of H7 antigen
Cultivate Escherichia coli O 157: H7, the centrifugal 30min of 3500r/min, resuspended with PBS, 0.5% formalin-inactivated, PBS are washed 3 times, and precipitation is resuspended in PBS, and-20 ℃ are frozen standby;
2) preparation of immune colloid gold
Get 20nm particle colloidal gold solution, add the sheep anti-mouse igg of purifying, at room temperature stir 30min, adding mass volume ratio 10% bovine serum albumin(BSA) BSA, to make its final concentration be mass volume ratio 0.4%, stirring at room 5min, adding mass volume ratio 10%PEG, to make the PEG final concentration be mass volume ratio 0.2%, stirring at room 5min; The centrifugal 45min of 12000r/min draws centrifugal supernatant, and precipitation is dissolved in 2ml preserves in the liquid, and with 0.45 μ m membrane filtration, it is standby to put 4 ℃ of preservations, makes colloid gold label antibody stoste;
3) preparation of the plain film of golden labeling antibody glass fibre
Get colloid gold label antibody stoste 1ml, add working fluid 2ml dilution, mixing is added on the plain film of glass fibre equably, makes the plain film of golden labeling antibody glass fibre, i.e. collaurum pad, and 37 ℃ of dryings, 4 ℃ of preservations are standby;
4) preparation of solid phase nitrocellulose filter NC
PBS with 0.01M pH7.2 dilutes Escherichia coli O 157 respectively: the mouse serum IgG of H7 antigen and purifying, with protein concentration is the 2.0mg/ml Escherichia coli O 157: the mouse serum IgG point sample of H7 antigen and purifying on the NC film respectively as detection line and nature controlling line, drying at room temperature, sealing, the chromatography test background is clear;
5) assembling of test strips
On wide by 2 * long 9cm PVC base plate (1), interlude is pasted wide by 2 * long 3cm solid phase nitrocellulose filter, i.e. NC film (2); Paste collaurum-sheep anti-mouse igg antibody bond glass fibre membrane (5) of 2 * 2cm near the PVC backplate surface of solid phase cellulose membrane detection line T line one end, plain film of this glass fibre and the overlapping 0.1cm of NC film (2), the overlapping 0.2cm of absorption of sample pad (7) of the other end and 2 * 1cm; The PVC backplate surface of close solid phase cellulose membrane nature controlling line C line one end is pasted the thieving paper (6) of 2 * 3.5cm, with the overlapping 0.1cm of NC film; Antibody solid phase nitrocellulose filter NC film (2) is gone up to draw an Escherichia coli O 157: a H7 Detection of antigen line T line (3) and a mouse serum IgG nature controlling line C line (4).
The T line is drawn on the position of long 3cm * wide 2cm NC film one side 1cm, with concentration is the Escherichia coli O 157 of 2.0mg/mL: H7 antigen is packed into and is drawn the film machine, be sprayed on the NC film, another shower nozzle is drawn nature controlling line C line, concentration is the mouse serum IgG of 2.0mg/mL, the C line is drawn on the position of NC film opposite side 1cm, the distance of detection line and nature controlling line is 1cm, the point sample amount is 5 μ l and is sprayed on the nitrocellulose membrane, the cardboard that assembles is by longitudinal shear, be cut into the strip that width is 2.5mm, be antibody test test strips finished product.
3, the usage of claim 1 or 2 described test strip is characterized in that:
When detecting antibody, mouse serum sample to be checked is diluted 100 times with the sample diluting liquid that contains multi-joint bacterial immunity rabbit anteserum, get 200 μ l and drip reaction zone in test strips, if red stripes all occurs at detection line place and nature controlling line place, show and contain Escherichia coli O 157 in the serum sample to be checked: H7 antibody is judged to the positive; If only red stripes occurs at the nature controlling line place, show not contain Escherichia coli O 157 in the sample to be checked: H7 antibody then is judged to feminine gender; If red stripes does not appear in nature controlling line,, represent that all the failure of an experiment or test strips lost efficacy no matter whether detection line red stripes occurs.
4, according to the usage of the described test strip of claim 3, it is characterized in that: use the PBS solution dilution of the 0.01M pH7.2 that contains volume ratio 2% multi-joint bacterial immunity rabbit anteserum to be tried the mouse serum sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910181759A CN101666799B (en) | 2009-07-23 | 2009-07-23 | Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910181759A CN101666799B (en) | 2009-07-23 | 2009-07-23 | Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101666799A true CN101666799A (en) | 2010-03-10 |
| CN101666799B CN101666799B (en) | 2012-10-10 |
Family
ID=41803510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910181759A Expired - Fee Related CN101666799B (en) | 2009-07-23 | 2009-07-23 | Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101666799B (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949925A (en) * | 2010-10-22 | 2011-01-19 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method |
| CN101957376A (en) * | 2010-10-22 | 2011-01-26 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for rapidly detecting guanidineacetic acid and derivatives thereof and preparation method thereof |
| CN101980021A (en) * | 2010-10-22 | 2011-02-23 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test strip for quickly detecting sibutramine and derivative thereof and preparation method |
| CN101986156A (en) * | 2010-10-22 | 2011-03-16 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for detecting glibenclamide and derivatives of glibenclamide quickly and preparation method thereof |
| CN102135538A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of escherichia coli O157:H7 and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN103792362A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Immune colloidal gold rapid detection test strip for enterohemorrhagic E.col O157:H7 |
| CN104251905A (en) * | 2014-09-01 | 2014-12-31 | 中国农业科学院兰州兽医研究所 | Haemophilus parasuis test paper strip and preparation method thereof |
| CN108061799A (en) * | 2017-12-07 | 2018-05-22 | 深圳市国赛生物技术有限公司 | A kind of chromatographic test paper strip device |
| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| CN111596076A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting sheep fertility |
| CN111948388A (en) * | 2020-08-18 | 2020-11-17 | 山东农业大学 | Colloidal gold test strip for detecting clostridium putrefactive, preparation method and application thereof |
| CN112698036A (en) * | 2020-12-22 | 2021-04-23 | 上海理工大学 | Immune test paper strip for rapidly determining cross reaction of bacterial monoclonal antibody and determination method |
| CN113203861A (en) * | 2021-05-12 | 2021-08-03 | 上海理工大学 | Visual antigen immunochromatographic test paper and determination method thereof |
| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
| CN115718196A (en) * | 2022-11-24 | 2023-02-28 | 上海交通大学医学院附属瑞金医院 | Bacterium multi-linked antigen detection test strip and application thereof |
-
2009
- 2009-07-23 CN CN200910181759A patent/CN101666799B/en not_active Expired - Fee Related
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102135538A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of escherichia coli O157:H7 and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| CN101957376A (en) * | 2010-10-22 | 2011-01-26 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for rapidly detecting guanidineacetic acid and derivatives thereof and preparation method thereof |
| CN101980021A (en) * | 2010-10-22 | 2011-02-23 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test strip for quickly detecting sibutramine and derivative thereof and preparation method |
| CN101986156A (en) * | 2010-10-22 | 2011-03-16 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for detecting glibenclamide and derivatives of glibenclamide quickly and preparation method thereof |
| CN101949925B (en) * | 2010-10-22 | 2013-07-31 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method |
| CN101949925A (en) * | 2010-10-22 | 2011-01-19 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method |
| CN103439496B (en) * | 2013-08-13 | 2015-04-15 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN103792362B (en) * | 2014-01-22 | 2015-10-07 | 上海理工大学 | Enterohemorrhagic Escherichia coli O 157: H7 immune colloid gold Rapid detection test strip |
| CN103792362A (en) * | 2014-01-22 | 2014-05-14 | 上海理工大学 | Immune colloidal gold rapid detection test strip for enterohemorrhagic E.col O157:H7 |
| CN104251905A (en) * | 2014-09-01 | 2014-12-31 | 中国农业科学院兰州兽医研究所 | Haemophilus parasuis test paper strip and preparation method thereof |
| CN108061799A (en) * | 2017-12-07 | 2018-05-22 | 深圳市国赛生物技术有限公司 | A kind of chromatographic test paper strip device |
| CN111521827A (en) * | 2020-05-21 | 2020-08-11 | 上海理工大学 | Immunofluorescence test strip for rapidly determining titer of monoclonal antibody based on antigen tracing |
| CN111596076A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting sheep fertility |
| CN111596076B (en) * | 2020-06-04 | 2023-05-02 | 昆明天沃生物科技有限公司 | Method for detecting sheep fertility |
| CN111948388A (en) * | 2020-08-18 | 2020-11-17 | 山东农业大学 | Colloidal gold test strip for detecting clostridium putrefactive, preparation method and application thereof |
| CN112698036A (en) * | 2020-12-22 | 2021-04-23 | 上海理工大学 | Immune test paper strip for rapidly determining cross reaction of bacterial monoclonal antibody and determination method |
| CN113203861A (en) * | 2021-05-12 | 2021-08-03 | 上海理工大学 | Visual antigen immunochromatographic test paper and determination method thereof |
| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
| CN115718196A (en) * | 2022-11-24 | 2023-02-28 | 上海交通大学医学院附属瑞金医院 | Bacterium multi-linked antigen detection test strip and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101666799B (en) | 2012-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101666799B (en) | Test strip for detecting colibacillus O157:H7 mouse serum antibody by colloidal gold immunochromatography | |
| CN104007261B (en) | Fowl three kinds of breathing problem three quick detection kit and application | |
| CN103439496B (en) | Escherichia coli O157:H7 enrichment and rapid detection method | |
| CN102393455B (en) | Preparation method of immunogold rapid test paper for vibrio parahaemolyticus | |
| CN101568836A (en) | Immobilisation and application of antigenic carbohydrates to detect infective micro-organisms | |
| CN104007265B (en) | Enterohemorrhagic Escherichia coli O 157: H7 quantum dot fluorescence immunochromatographiassay assay quantitative detection test paper and detection method thereof | |
| CN101203760A (en) | Immobilisation of antigenic carbohydrates to support detection of pathogenic micro-organisms. | |
| CN101358979B (en) | Chloramphenicol immune detecting system marked by magnetic bead | |
| CN103439497A (en) | Salmonella enrichment and rapid detection method | |
| CN104181298B (en) | A kind of human parainfluenza virus's IgM antibody test strip and preparation method thereof | |
| CN102818898A (en) | Test strip for identifying foot-and-mouth-disease virus infected and vaccine immunized animal at one step and preparation method of test strip | |
| CN103743908A (en) | Neomycin semi-quantitative gold-labeled rapid detection kit, and detection method and application thereof | |
| CN101109753A (en) | Method for detecting colon bacillus shiga toxin with colloidal gold immune chromatography experiment | |
| CN104374914A (en) | Pseudomonas putida test strip and preparation method thereof | |
| CN106932575A (en) | The method of nano immune magnetic bead technical tie-up Enzyme-linked Immunosorbent Assay quick detection salmonella typhimurium | |
| CN101082059A (en) | Bacillus coli O157:H7 antigen rapid detection testing strip | |
| CN102135538A (en) | Detection method of escherichia coli O157:H7 and gold-labeled rapid diagnosis kit for same and preparation method thereof | |
| CN105891412B (en) | A kind of citrobacter freundii test strip and preparation method thereof | |
| CN101943701A (en) | Preparation method of colloidal gold test strip for quickly detecting chloramphenicol residues | |
| CN101520459B (en) | Rabbit hemorrhagic disease virus colloidal gold test strip | |
| CN102033128B (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN103823061A (en) | Au/Fe3O4 immunity chromatography test paper, preparation method and applications thereof | |
| CN103698506A (en) | Method for detecting content of pollutants in sample by utilizing direct immuno-PCR (polymerase chain reaction) assay | |
| CN202599960U (en) | Colloidal gold test strip for rapid detection of O-type foot-and-mouth disease virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121010 Termination date: 20130723 |