CN101968487A - Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof - Google Patents
Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof Download PDFInfo
- Publication number
- CN101968487A CN101968487A CN200910206028XA CN200910206028A CN101968487A CN 101968487 A CN101968487 A CN 101968487A CN 200910206028X A CN200910206028X A CN 200910206028XA CN 200910206028 A CN200910206028 A CN 200910206028A CN 101968487 A CN101968487 A CN 101968487A
- Authority
- CN
- China
- Prior art keywords
- hemolysin
- listeria spp
- listeria
- test paper
- polyclonal antibodies
- 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.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to immune colloidal gold test paper for detecting listeria hemolysin and a preparation method thereof, belonging to the field of biotechnology. In the invention, firstly, the listeria hemolysin expressed by the recombinant Escherichia coli is purified, and then, the purified listeria hemolysin is used for preparing polyclonal antibodies and monoclonal antibodies. Colloidal gold is used for marking the monoclonal antibodies as tracers, anti-hemolysin polyclonal antibodies and anti-mouse polyclonal antibodies are respectively coated in the corresponding positions of a detection line and a quality control line, and the anti-hemolysin polyclonal antibodies and the anti-mouse polyclonal antibodies can be specifically combined with corresponding antigens to be trapped and reddened. The negative and positive results can be judged according to whether the detection line and the quality control line develop or not. The invention has the characteristics of simple and convenient sampling, strong specificity, high sensibility and the like, can conveniently, quickly and accurately detect the listeria hemolysin in 5min, and can also be used for detecting whether samples of animals, animal tissues, silage, foods, meat products, excrement and the like are polluted by the listeria capable of generating the hemolysin.
Description
(1) technical field
The present invention relates to a kind of immune colloid gold test paper and detection method thereof that detects the listeria spp hemolysin, also relate to the preparation method of this test paper related reagent simultaneously.
(2) technical background
Listeria spp (Listeria) is distributed widely in the environment, comprise altogether Ge Shi listeria spp (L.grayi), harmless listeria spp (L.innocua), Yi Shi listeria spp (L.ivanovii), monocyte hyperplasia listeria spp (Listeria Monocytogenes, LMO), Mo Shi listeria spp (L.murrayi), Si Shi listeria spp (L.seeligeri), seven kinds of Wei Shi listeria spp (L.welshimeri).
It is the monocyte hyperplasia listeria spp that listeria spp belongs to representative species, and it is a kind of pathogenic bacteria that can cause zoonosis and food origin disease.LMO is distributed widely in nature, as is prevalent in the environment of agricultural product, ensilage, mire and wetland and preservation and processing food.This bacterium is extremely strong to the environment resistibility, can both well-grown under the situation of low temperature (4 ℃), high temperature (42 ℃), sour environment, low oxygen concentration and high salt (20%) and produce pathogenicity.Therefore in normal food, food processing environment and domestic refrigerator, can survive the long duration.LMO not only often appears at living animal or plant food, ensilage, all can find its trace at many prepared food or processed food in as the fish of cold sterilization milk, cheese and ice cream, sootiness or meat product.This Pseudomonas is in endotrophic Gram-positive bacillus, confirmed at present to infect more than 40 kind of animal, behind the human poultry infection, mainly shown as symptoms such as meningitis, septicemia and blood monocytes increase, mortality ratio reaches 20%-30%, and Developing of Animal Industry has been caused very serious harm.Be present in the food poisoning that can cause the people in the food and produce the listeria spp hemolysin, can cause bacteremia and meningitis, can cause miscarriage, the health of humans and animals has been constituted serious threat the pregnant woman.Detecting LMO method commonly used at present mainly comprises: traditional micro-biological process, immunofluorescence technique, enzyme linked immunosorbent assay (ELISA), PCR and nucleic acid probe detection technique.Traditional micro-biological process is that the suspicious bacterium colony after the separation pure culture is carried out evaluations such as biochemical reaction, zoopery, typical motion, is confirmed as the clear type analysis of the laggard stepping promoting circulation of blood of LMO.This method is a method in common comparatively at present, be characterized in accurate, simple to operate, but need the time longer, and resultant error is bigger, and the manpower and materials cost is bigger, the needs of fast detecting in the incompatibility practical application.The Chang Yinwei fluorescence antibody is stable inadequately in the immunofluorescence technique, and judges subjectively, is prone to cross reaction, so many limitation are arranged when using.The ELISA method widely is used as the method for quick of qualitative or half-quantitative detection in recent years, though shortened detection time to a certain extent, but still need special instrument (as microplate reader), operating process is still complicated.PCR and nucleic acid probe detection technique are highly sensitive, reliable results, but assay method is loaded down with trivial details, time-consuming, and efficient is low, cost is high, often is subjected to the interference of food substrate, medium component and assorted bacterium, and operating personnel need pass through professional training, are difficult to popularize.These methods must be done more biochemical test or zoopery and could differentiate the pathogenicity Listeria to come out from sample.More main is that it is higher that above detection method is the laboratory degree of dependence, needs special equipment, and operating personnel's professional skill is had relatively high expectations, and limited and promoted the use of.
Listeria hemolysin (Listeriolysin O, LLO), molecular weight by the hly gene code is the cytolysin albumen of 58.6kDa, belong to cell hemolysin family in conjunction with cholesterol activation sulfydryl, it is the main virulence factor of LMO secretion, its disappearance will cause the total loss of bacterial virulence, and it can damage red blood cell, macrophage (cracking lysosome membrane and phagosome film) and blood platelet.Can in nonphagocytic cytosol, not survive a period of time though do not produce the LMO mutant strain of LLO, irreproducible, and because of the phagosome of can't escaping can not make other cell infections, in addition, LLO has also participated in pathogenic other relevant reaction with LMO.
Except that LMO, Yi Shi listeria spp and Si Shi listeria spp also can be secreted LLO.
Immunochromatography technique is a kind of simple and easy technology of immunology detection fast that development in recent years is got up, be with the collaurum be the tachysynthesis of label in conjunction with test, antigen, antibody react on the NC film.This technology have high specificity, highly sensitive, good stability, simple and easy quick, easy and simple to handle, quick, visual result, need not instrument and equipment etc. advantage, also not needing to carry out professional training can operate, alleviate labor intensity of operating personnel simultaneously, have widespread use and be worth.
The present invention is with the distinctive significant virulence albumen of listeria spp---listeria spp hemolysin (Listeriolysin O, LLO) as detected object, the toxin that exists in can test sample, also can be used for the detection of listeria spp, and can overcome in the classic method with pathogen thalline constituent as cross reaction that detected object was occurred is taken place with other Related Bacteria, simultaneously avoided the animal toxicity test sense cycle long as detected object with virulence marker gene and product thereof, animal individual difference is big, drawbacks such as unstable result.Association colloid gold immunochromatography technique makes detection method of the present invention more special, quick, with strong points, and is practical and convenient.
(3) summary of the invention
The invention provides immune colloid gold test paper of listeria spp hemolysin in a kind of test sample and preparation method thereof.This test strips is by sample pad, the collaurum pad, and nitrocellulose filter, thieving paper and PVC base plate are formed.PVC backing one end is stained with sample pad successively, the collaurum pad, and the attached nitrocellulose filter of pars intermedia, the other end is stained with thieving paper.Between described sample pad and the described collaurum pad overlap joint is arranged, between described nitrocellulose filter and the collaurum pad overlap joint is arranged; Between described nitrocellulose filter and the thieving paper overlap joint is arranged; Described collaurum pad is made up of the plain film of the glass fibre of the antihemolysin monoclonal antibody that is adsorbed with colloid gold label, is coated with the nature controlling line that antihemolysin detection line how grand antibody constitutes and rabbit anti-mouse igg constitute on the described nitrocellulose filter respectively.
Be used to immunity colloidal gold test paper strip that detects LLO and preparation method thereof, filling up does not have the blank that detects the LLO immune colloidal gold detection test paper strip at present as yet and utilizes detection LLO virulence factor to detect the blank of the immune colloidal gold detection test paper strip of LMO.The present invention has following advantage:
1, the immunity colloidal gold test paper strip of detection LLO of the present invention has high specificity, and is highly sensitive, lacks advantages such as (in 5 minutes) detection time.
2, test strip of the present invention does not need specific apparatus, equipment, and technology is simple, and it is low to detect cost.
3, use the present invention from sample, to detect LLO, can be used as and contain the foundation of producing the hemolysin listeria spp in the sample, can avoid using the step that needs to determine virulence in the existing detection method with animal experiment.
The preparation method of this test paper may further comprise the steps:
1) reorganization LLO antigen preparation
With the recombination bacillus coli abduction delivering, behind SDS-PAGE electrophoretic analysis purpose band, purifying protein carries out SDS-PAGE and analyzes purification result, measures protein concentration earlier.
2) anti-LLO MONOCLONAL ANTIBODIES SPECIFIC FOR
With the LLO albumen of purifying, divide immune BalB/c mouse 3 times, the spleen of getting immunity back mouse carries out Fusion of Cells, after sieving through three subclones, obtains 6 strain monoclonal antibodies altogether through western blotting, identified by immunofluorescence.This 6 strain monoclonal antibody after measured, specificity height, secretion are stable.The hybridoma cell strain IIC4 that choose wherein that a strain secretory antibody subclass is IgG1, tires the highest is deposited in Chinese typical culture collection center.
2) a large amount of preparations and the purification of anti-LLO monoclonal antibody
Hybridoma cell strain IIC4 is injected in the mouse peritoneal of sensitization, treated that mouse web portion obviously swelled collection ascites in 12～15 days, centrifugal, can obtain a large amount of monoclonal antibodies.Ascites must be through sad-saturated ammonium sulfate method by classification extracting, PBS dialysis, centrifugal, and through proteinG affinity column purifying, antibody purity reaches 95% after measured again.
3) anti-LLO Polyclonal Antibody Preparation
With the LLO albumen of purifying, take antiserum after dividing 3 immunizing rabbits.The polyclonal antibody monoclonal antibody is through sad-saturated ammonium sulfate method runic, and through proteinA affinity column purifying, antibody purity reaches 95% after measured again.
4) preparation of goat anti-mouse IgG polyclonal serum
With the mouse IgG immune goat of purifying, after the acquisition hyper-immune serum, purifying obtains goat anti-mouse IgG polyclonal serum (mountain sheep anti mouse two is anti-).
5) preparation of colloidal gold probe:
At first adopt trisodium citrate reduction method to prepare the collaurum of diameter 20nm,, carry out with particular solution the precipitation after centrifugal being hanged after the high speed centrifugation afterwards with the anti-LLO monoclonal antibody of 0.9mg/ml ratio adding purifying.
6) preparation of collaurum pad:
Colloidal gold probe is uniformly coated on the glass fibre, and at room temperature the collaurum pad is made in oven dry.
7) will dilute good antihemolysin polyclonal antibody, mountain sheep anti mouse two anti-detection zone and the Quality Control districts that are coated on respectively on the nitrocellulose filter, two line interval 0.5cm, drying at room temperature after the spraying with the diluted liquid of specified packet.
8) shown in figure one, assemble test strips, paste sample pad, collaurum pad, nitrocellulose filter and thieving paper on the PVC base plate successively.The test strips of having assembled is cut into the width of 3mm.
9) detect
If when sample to be checked is liquid substance such as water, milk, bacterial cultures, serum, can directly get 50 μ l sample surveys.When if sample to be checked is sample such as animal and tissue thereof, ensilage, food, meat products, ight soil, then need sample is carried out pre-service, being about to sample joins in BHI (or TSB-YE) nutrient culture media, after 37 ℃ of shaking table 230r/min cultivate 10h, the centrifugal 15min of 5000r/min gets supernatant 50 μ L and detects usefulness.
Testing result: red purpose band appears in detection zone, and is then positive, shows to contain hemolysin in the sample or have the listeria spp that produces hemolysin.
The Figure of description explanation:
Accompanying drawing 1 is test strips front elevation and positive each section explanation.
1.PVC base plate, 2. thieving paper, 3. nature controlling line, 4. detection line, 5. nitrocellulose filter, 6. the collaurum pad, 7. sample pad accompanying drawing 2 is test strips side-looking structural drawing and each section explanation.
1.PVC base plate, 2. thieving paper, 3. nature controlling line, 4. detection line, 5. nitrocellulose filter, 6. the collaurum pad, 7. sample pad accompanying drawing 3 is the explanation of test strips testing result.
The preservation explanation:
Preservation date: on July 11st, 2009
Depositary institution's full name: Chinese typical culture collection center
Depositary institution is called for short: CCTCC
Depositary institution address: Wuhan City Wuhan University, 430072
Classification name: the hybridoma cell strain II C4 that secretes anti-listeria spp hemolysin monoclonal antibody
Preserving number: CCTCC NO:C200917
Embodiment
Embodiment one reorganization LLO antigen preparation
Earlier recombination bacillus coli pGEX-6P-hly/BL21 is inoculated in the fluid nutrient medium (containing ampicillin 100 μ g/mL), 230r/m cultivates the 12h activation for 37 ℃.To activate in thalline is inoculated in fluid nutrient medium in 1: 100 ratio the triangular flask (containing ampicillin 100 μ g/ml), 230r/m, 37 ℃ of cultivations add IPTG and induce when waiting to be cultured to 150min, its final concentration is 0.6mmol/L, finishes abduction delivering when continuing to cultivate 150min.After SDS-PAGE electrophoretic analysis purpose band is expressed, carry out going up in a large number sample, with the dyeing of KCl dye liquor, the purpose band is downcut, pulverize the back and use the PBS multigelation, albumen is dissolved in wherein, the micelle of multigelation is centrifugal, it is an amount of to get supernatant, add equivalent SDS sample-loading buffer, fully mixing boiled 5 minutes, to induce the back thalline is contrast, carries out the electrophoretic analysis purification result on 12%SDS-PAGE.
The preparation of embodiment two immune colloid gold test papers
PVC is done backing, above an end be stained with thieving paper successively, glass fibre membrane, nitrocellulose filter is sticked in the centre, the other end is stained with water-absorption fiber.Wherein adsorbing the monoclonal antibody of gold mark hemolysin on the glass fibre membrane, detection zone is coated with the polyclonal antibody of antihemolysin on the nitrocellulose filter, and wrap by the polyclonal antibody of sheep anti-mouse igg in the Quality Control district.
Preparation according to the following steps:
(1) anti-LLO MONOCLONAL ANTIBODIES SPECIFIC FOR: with purification of Recombinant LLO albumen, divide 3 immune Balb/C (ATCC CRL 1772) mouse, the spleen of getting immunity back mouse carries out Fusion of Cells, obtains 6 strain monoclonal antibodies altogether through western-blotting, identified by immunofluorescence.This 6 strain monoclonal antibody after measured, specificity height, secretion are stable.The hybridoma cell strain II C4 that choose wherein that a strain secretory antibody subclass is IgG1, tires the highest is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C200917.
(2) a large amount of preparations and the purification of monoclonal antibody:
Get some 6～8 age in week healthy BABL/c mouse, lumbar injection 0.5ml/ only sterilizes paraffin oil as sensitizing agent, injects 1 * 10 to the abdominal cavity again after 10 days
5Individual well-grown hybridoma II C4, mouse web portion swelling after 12～15 days, collect ascites, with 4 ℃, the centrifugal 15min of 3000r/min, get centrifugal back supernatant filtering with microporous membrane, remove bigger grumeleuse and fat drop, remove cell residue and finely ground particle substance with 4 ℃, the centrifugal 15min of 10000r/min again.
At first adopt sad-saturated ammonium sulfate method that ascites is slightly carried, method of operating is as follows: the acetate buffer (0.06mol/L that adds 2ml in 1ml ascites, PH5.0), transfer PH to 4.5 with hydrochloric acid, it is sad dropwise to add 33 μ l under stirring at room in 30min, 4 ℃ leave standstill 2h, the centrifugal 30min of 12000r/min, abandon precipitation and get supernatant, 0.1mol/LPB (the PH7.4 that in supernatant, adds 1/10 volume, 8.5%NaCl), and transfer PH to 7.4, under 4 ℃ of ice baths, in 30min, add the ammonium sulfate of 0.277g/ml with the NaOH of 1mol/L, make into 45% saturation degree, leave standstill more than the 1h, 4 ℃ of centrifugal 30min of following 12000r/min abandon supernatant, precipitation is dissolved in 0.01mol/L PB, PH7.0, to 100 times above-mentioned PB in 4 ℃ of dialysed overnight, until can not detecting NH4 with Nai Shi reagent and 0.5mol/L barium chloride solution
+And SO4
2-Adopt Protein G (GEHealthcare company) affinity chromatography method to be further purified monoclonal antibody then.
(3) anti-LLO Polyclonal Antibody Preparation of rabbit and purifying
With the hemolysin albumen and the emulsification of adjuvant mixed in equal amounts of purifying, initial immunity Freund's complete adjuvant (CFA) emulsification, every 7 days booster immunizations once, booster immunization incomplete Freund (IFA) emulsification.Vaccination ways is selected 6 inoculations of subcutaneous back portion.The ear vein blood sampling once after ELISA detection antibody titer reaches desired level, is carried out the heart blood sampling week about after immunity.37 ℃ of institute's blood samplings are placed 30min, and 4 ℃ are spent the night, second day 4 ℃, 3000r/min, centrifugal 15min, getting supernatant is polyclonal antiserum.At first adopt sad-saturated ammonium sulfate method that serum is slightly carried during the polyclonal antibody purifying.Acetate buffer (the 0.06mol/L that in 1ml serum, adds 2ml, pH5.0), transfer pH to 4.5 with hydrochloric acid, it is sad dropwise to add 75 μ l under stirring at room in 30 minutes, 4 ℃ left standstill 2 hours, centrifugal 30 minutes of 12000r/min, abandon precipitation and get supernatant, the pH7.4 that in supernatant, adds 1/10 volume, 0.1mol/L PB, 8.5%NaCl, and transfer pH to 7.4 with the NaOH of 1mol/L, the ammonium sulfate that under 4 ℃ of ice baths, in 30min, adds 0.277g/ml, make into 45% saturation degree, leave standstill more than the 1h, then 4 ℃ of centrifugal 30min of 12000r/min, abandon supernatant, precipitation is dissolved among the pH7.80.01mol/L PB, to 100 times pH7.8,0.1mol/L PB is in 4 ℃ of dialysed overnight, until can not detecting NH with Nai Shi reagent and 0.5mol/L barium chloride solution
4 +And SO
4 2-How anti-adopt albumin A (GEHealthcare company) affinity chromatography method to be further purified then.
(4) Polyclonal Antibody Preparation of mountain sheep anti-mouse igg and purifying
Purification mouse IgG, as immunogene and the emulsification of adjuvant mixed in equal amounts, initial immunity Freund's complete adjuvant (CFA) emulsification, every 14 days booster immunizations once, booster immunization incomplete Freund (IFA) emulsification.Vaccination ways is selected intramuscular inoculation.The jugular vein blood sampling once after ELISA detection antibody titer reaches desired level, is carried out the jugular vein blood sampling week about after immunity.37 ℃ of institute's blood samplings are placed 30min, and 4 ℃ are spent the night, second day 4 ℃, 3000r/min, centrifugal 10min, getting supernatant is polyclonal antiserum.At first adopt sad-saturated ammonium sulfate method that serum is slightly carried during the purifying of polyclonal antibody.Acetate buffer (the 0.06mol/L that in every 1ml serum, adds 2ml, pH5.0), transfer pH to 4.5 with hydrochloric acid, it is sad dropwise to add 75 μ l under stirring at room in 30 minutes, 4 ℃ left standstill 2 hours, centrifugal 30 minutes of 12000r/min, abandon precipitation and get supernatant, the pH7.4 that in supernatant, adds 1/10 volume, 0.1mol/L PB, 8.5%NaCl, and transfer pH to 7.4 with the NaOH of 1mol/L, the ammonium sulfate that under 4 ℃ of ice baths, in 30min, adds 0.277g/ml, make into 45% saturation degree, leave standstill more than the 1h, then 4 ℃ of centrifugal 30min of 12000r/min, abandon supernatant, precipitation is dissolved among the pH7.8 0.01mol/L PB, to 100 times pH7.8,0.1mol/L PB is in 4 ℃ of dialysed overnight, until can not detecting NH with Nai Shi reagent and 0.5mol/L barium chloride solution
4 +And SO
4 2-How anti-adopt albumin A (GEHealthcare company) affinity chromatography method to be further purified then.
(5) preparation of colloidal gold probe
Adopt the sodium citrate reducing process to prepare collaurum: all glasswares to be cleaned up, be immersed in 24h in the washing lotion (adding distil water is to 10000ml for potassium dichromate 1000g, concentrated sulphuric acid 2500ml) then.Take out the back with tap water flushing 8 times, distilled water flushing 3 times after the oven dry is soaked in glass container 1min in the chloroformic solution of 5% dichloro dipotassium silane, uses deionized water rinsing then, again drying for standby.Add 0.01% chlorauric acid solution 100ml, agitating heating on magnetic stirring apparatus, add 2.5ml 1% sodium citrate rapidly, continue heated and boiled 5～8min, solution colour is transferred to orange red gradually by mazarine, lovely luster stops to boil, and be back to original volume 100ml, 4 ℃ airtight keep in Dark Place standby.
K with 0.1mol/L
2CO
3The pH value of colloidal gold solution is transferred to pH8.2, transfer to slowly concussion on the shaking table in the lucifuge condition, the monoclonal antibody of getting purifying dropwise is added to final concentration 0.9mg/ml, continue concussion 30min, leave standstill 10min, add BSA as stabilizing agent, make its final concentration reach 1%, slowly shake 30min again.
Gold is marked monoclonal antibody solution remove collaurum polymkeric substance wherein with 4 ℃, the centrifugal 10min of 2000r/min, get supernatant then, again with 4 ℃, the centrifugal 60min of 12000r/min, as seen centrifugal thing is divided into 3 layers, supernatant contains still unconjugated monoclonal antibody, and nethermost aterrimus spot is still unconjugated colloid gold particle polymkeric substance.The centre is mauve flocculent deposit and is in conjunction with good gold mark monoclonal antibody compound.Collect the aubergine flocculent deposit, with 10mmol/LpH8.2PB (the containing 1%BSA) suspension of original volume, 4 ℃ keep in Dark Place standby.
(6) the collaurum pad prepares the preparation of glass fibre membrane probe
Earlier glass fibre membrane is immersed in 30min in 0.02mol/L Tris-HCl (pH8.2) solution that contains 3%BSA, 0.1%Tween-20,4 ℃ of dried overnight are standby.Amount by every glass fibre membrane (size of glass fibre membrane is 0.7cm * 7cm/ bar) 0.7ml colloidal gold probe evenly is sprayed at colloidal gold probe on the glass fibre, at room temperature oven dry.
(7) antihemolysin polyclonal antibody, mountain sheep anti-mouse igg two anti-concentration (wrapping the TBS that diluted liquid is 0.01mol/L pH8.2) with 1mg/ml are coated on nitrocellulose filter (HF135NC film, Millipore company) detection zone on and Quality Control district, article two, line interval 0.5cm wraps quilt drying at room temperature afterwards.In the 0.02mol/LTris-HCl that contains 1%BSA, 0.2%Tween-20,0.05mol/L (pH8.4) solution, room temperature is sealed 10-60min with the nitrocellulose filter behind the bag quilt; Afterwards, use 0.05mol/LNaHPO
4After twice of the rinsing, 4 ℃ of drying for standby.
(8) will wrap by good nitrocellulose filter and adhere on the PVC base plate, notice that the lower edge of film aligns floating face with density bullet line on the mould.The glass fibre of the good golden labeling antibody of spraying is adhered on the PVC base plate near the scale lower limb, floating.Sample pad is adhered on the PVC base plate near the mould lower limb, floating.Thieving paper is adhered on the PVC base plate near the mould coboundary, floating.Be cut into the wide test paper of 3mm with cutting cutter, the test paper that cuts is put into the packaging bag that drying agent is housed, standby.
Embodiment three test strips detect
1. test strips is used and is judged:
(1) if when sample to be checked is liquid substance such as water, milk, bacterial liquid culture, serum, can directly get 50 μ l sample surveys.When if sample to be checked is sample such as animal and tissue thereof, ensilage, food, meat products, ight soil, then need sample is carried out pre-service, being about to sample joins in BHI (or TSB-YE) nutrient culture media, after 37 ℃ of shaking table 230r/min cultivate 10h, the centrifugal 15min of 5000r/min gets supernatant 50 μ L and detects usefulness.
(2) detect: getting liquid is dripped on the sample pad, act on 3～5min under the room temperature.
(3) result judges:
Positive findings, nature controlling line, detection line all present red stripes, have illustrated that the listeria spp hemolysin exists.
Red stripes does not appear in negative findings, detection line, and nature controlling line presents red stripes, and illustrating does not have monokaryon hyperplasia Li bacillus to exist in the sample.
If detection line, nature controlling line red stripes all do not occur or only have detection line red stripes to occur, product failure is described then.
2. specificity test
The bacterial strain of relevant generation hemolysins such as megacoccus, streptococcus, erysipelas bacillus, staphylococcus aureus, C.perfringens, clostridium tetani is detected, the result shows that the test paper of preparation with megacoccus, streptococcus, erysipelas bacillus, staphylococcus aureus, C.perfringens, clostridium tetani etc. cross reaction does not take place.
3. sensitivity tests
With PBS preparation LLO standard items gradient solution, be respectively 0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, with three batches of the detection paper of the present invention's preparation, each concentration is set 3 repetitions, and the result confirms, is limited to 20ng/ml under detecting.
Table 1 test strips sensitivity tests result
4. the accuracy test of test strips
Water, milk, bacterial liquid culture, serum etc. are confirmed as interpolation LLO standard items in the negative sample, its final concentration is respectively is 0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, detect then, each sample triplicate, testing result and standard items testing result 100% meet.Concrete outcome sees Table 2.
The accuracy test result of table 2 test strips
5. repeatable
Test paper with polybag and foil sealing, is added a drying agent part and places 4 ℃, and a part places 37 ℃ of preservations, detects once week about, whenever establishes three repetitions, detects its stability.The result shows that 4 ℃ of placement 120d, 37 ℃ of placement 20d still can detect the positive.
Four couples of embodiment contain the analog sample that produces the hemolysin listeria spp and detect
The analog samples such as water sample, milk sample, raw meat goods, vegetables, freezing shrimp meat and bread that are added with listeria spp are detected, sample thief is inoculated in BHI (or TSB-YE) nutrient culture media, after 37 ℃ of shaking table 230r/min cultivate 10h, the centrifugal 15min of 5000r/min gets supernatant 50 μ l and detects usefulness.The result shows, is 4.2 * 10 when analog sample adds bacterial concentration
9CFU/mL, 4.2 * 10
8CFU/mL, 4.2 * 10
7The sample detection result of CFU/mL all is positive, and is 4.2 * 10 and add bacterial concentration
6CFU/mL, 4.2 * 10
5CFU/mL, 4.2 * 10
4CFU/mL and 4.2 * 10
3The sample of CFU/mL and negative control group testing result all are negative, and therefore, the sensitivity that starter bacteria in the analog sample is detected is 4.2 * 10
6CFU/mL.
Claims (4)
1. the immune colloid gold test paper of listeria spp hemolysin in the working sample comprises sample pad, collaurum pad, nitrocellulose filter, thieving paper and PVC base plate, it is characterized in that:
1) the collaurum pad is made of by the gold of listeria spp hemolysin mark monoclonal antibody bag on glass fibre membrane, and the detection zone bag is by the polyclonal antibody of anti-listeria spp hemolysin on nitrocellulose membrane;
2) bag of the Quality Control district on nitrocellulose membrane is by the polyclonal antibody of anti-mouse IgG.
2. the gold mark monoclonal antibody of listeria spp hemolysin according to claim 1 is characterized in that: use Bacillus coli expression reorganization listeria spp hemolysin as immunogene, immune BALB/c mouse.The splenocyte and the murine myeloma cell sp/20 that get immunity back mouse carry out Fusion of Cells, after three subclone screenings, obtain the hybridoma of secretion at monocyte hyperplasia listeria spp hemolysin monoclonal antibody through western blotting, identified by immunofluorescence.The secreted monoclonal antibody specificity of hybridoma height, good stability.
3. the polyclonal antibody of anti-listeria spp hemolysin according to claim 1 is characterized in that: the reorganization listeria spp hemolysin albumen with purifying prepares behind the immunizing rabbit.
4. the immune colloid gold test paper of listeria spp hemolysin according to claim 1, it is characterized in that being used for convenient, fast, detect the listeria spp hemolysin exactly, also can be used for detecting whether polluted the listeria spp that produces hemolysin in the samples such as animal and tissue thereof, ensilage, food, meat products, ight soil.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910206028XA CN101968487A (en) | 2009-03-31 | 2009-10-09 | Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910071680.5 | 2009-03-31 | ||
| CN200910071680 | 2009-03-31 | ||
| CN200910206028XA CN101968487A (en) | 2009-03-31 | 2009-10-09 | Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101968487A true CN101968487A (en) | 2011-02-09 |
Family
ID=43547672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910206028XA Pending CN101968487A (en) | 2009-03-31 | 2009-10-09 | Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101968487A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539770A (en) * | 2011-12-29 | 2012-07-04 | 何鑫 | Reagent kit for detecting antiuninuclear cell proliferation Listeria bacteria by colloidal gold Hly gene monoclonal antibodies |
| CN103454419A (en) * | 2013-07-22 | 2013-12-18 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
| CN103941003A (en) * | 2013-03-15 | 2014-07-23 | 河南省农业科学院 | Swine listeriosis, swine necrobacillosis and swine francisella tularensis tri-combination detection test paper strip |
| CN104165989A (en) * | 2014-07-10 | 2014-11-26 | 长春理工大学 | Colloidal gold immunization test paper strip for detecting morchella mycelium |
| CN106546742A (en) * | 2016-11-01 | 2017-03-29 | 甘肃省畜牧兽医研究所 | A kind of gold mark detection test paper bar based on goat capripoxviruses and preparation method thereof |
| CN114736295A (en) * | 2022-06-14 | 2022-07-12 | 北京科跃中楷生物技术有限公司 | Horseradish peroxidase labeled antibody and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1963521A (en) * | 2005-11-10 | 2007-05-16 | 北京庄笛浩禾生物医学科技有限公司 | Rapid test paper bar for testing colloidal gold of 01 group cholera vibrio |
-
2009
- 2009-10-09 CN CN200910206028XA patent/CN101968487A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1963521A (en) * | 2005-11-10 | 2007-05-16 | 北京庄笛浩禾生物医学科技有限公司 | Rapid test paper bar for testing colloidal gold of 01 group cholera vibrio |
Non-Patent Citations (2)
| Title |
|---|
| 秦玉敏: "《李氏溶血素单克隆抗体制备及检测李氏溶血素的初步探索》", 《中国优秀硕士学位论文全文数据库》 * |
| 董慧等: "《抗李斯特菌溶血素单克隆抗体的制备与初步鉴定》", 《细胞与分子免疫学杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539770A (en) * | 2011-12-29 | 2012-07-04 | 何鑫 | Reagent kit for detecting antiuninuclear cell proliferation Listeria bacteria by colloidal gold Hly gene monoclonal antibodies |
| CN103941003A (en) * | 2013-03-15 | 2014-07-23 | 河南省农业科学院 | Swine listeriosis, swine necrobacillosis and swine francisella tularensis tri-combination detection test paper strip |
| CN103941003B (en) * | 2013-03-15 | 2016-07-06 | 河南省农业科学院 | Pig Listeria monocytogenes, pig necrobacillus and pig Bacillus tularensis three test strip |
| CN103454419A (en) * | 2013-07-22 | 2013-12-18 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
| CN103454419B (en) * | 2013-07-22 | 2015-03-25 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
| CN104165989A (en) * | 2014-07-10 | 2014-11-26 | 长春理工大学 | Colloidal gold immunization test paper strip for detecting morchella mycelium |
| CN106546742A (en) * | 2016-11-01 | 2017-03-29 | 甘肃省畜牧兽医研究所 | A kind of gold mark detection test paper bar based on goat capripoxviruses and preparation method thereof |
| CN114736295A (en) * | 2022-06-14 | 2022-07-12 | 北京科跃中楷生物技术有限公司 | Horseradish peroxidase labeled antibody and preparation method thereof |
| CN114736295B (en) * | 2022-06-14 | 2022-08-09 | 北京科跃中楷生物技术有限公司 | Horseradish peroxidase labeled antibody and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101968487A (en) | Immune colloidal gold test paper for detecting listeria hemolysin and preparation method thereof | |
| CN103123354B (en) | Immunodetection assay for mycobacterium tuberculosis complex | |
| CN104316690B (en) | Vibrio parahemolyticus immune colloid gold Rapid detection test strip | |
| Seo et al. | An ELISA-on-a-chip biosensor system coupled with immunomagnetic separation for the detection of Vibrio parahaemolyticus within a single working day | |
| CN109655435A (en) | A kind of fluorescent quenching test paper and the preparation method and application thereof detecting okadaic acid | |
| CN103333864B (en) | Monoclonal antibody of toxoplasma gondii resistant MIC3 protein and application monoclonal antibody | |
| CN101613706A (en) | The colloidal gold labeling lap gene monoclonal antibody measuring Listeria monocytogenes kit | |
| CN102590527A (en) | Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof | |
| CN101887061A (en) | Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof | |
| CN103941020A (en) | Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting haemophilus parasuis antibody | |
| EP2972367A1 (en) | Biomarkers for detecting the presence of bacteria | |
| CN104991069B (en) | Acidovorax avenae subsp immune colloidal gold detection test paper strip and monoclonal antibody thereof and hybridoma cell strain | |
| CN104178458B (en) | One plant of Listeria monocytogenes monoclonal antibody hybridoma cell strain and its application | |
| CN105541977A (en) | Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof | |
| CN102590513A (en) | Immuo-colloidal gold test paper for detecting bacillus cereus and preparation method thereof | |
| CN102477097A (en) | Preparation method of monoclonal antibody to chloramphenicol | |
| CN105777900B (en) | Acidovorax avenae subsp monoclonal antibody and its hybridoma cell strain | |
| CN101942414B (en) | Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same | |
| CN103412122B (en) | Indirect competitive ELISA (Enzyme Linked Immunosorbent Assay) method based on multi-epitope tandem peptides and used for synchronously detecting staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin G (SEG) | |
| CN103792362B (en) | Enterohemorrhagic Escherichia coli O 157: H7 immune colloid gold Rapid detection test strip | |
| CN102539770A (en) | Reagent kit for detecting antiuninuclear cell proliferation Listeria bacteria by colloidal gold Hly gene monoclonal antibodies | |
| GENIGEQRGIS et al. | Immunofluorescent Detection of Staphylococcal Enterotoxin B II. Detection in Foods | |
| CN103941011B (en) | Detect method and the monoclonal antibody thereof of listeria monocytogenes | |
| Essery et al. | Detection of microbial surface antigens that bind Lewisa antigen | |
| CN105137075B (en) | Campylobacter jejuni immune colloid gold Rapid detection test strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110209 |