CN106093409A - The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody belongs to - Google Patents
The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody belongs to Download PDFInfo
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Abstract
The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody SQX6D8 belongs to, belongs to immunoassay field.The conjugate of the Salmonella typhimurium lipopolysaccharide (LPS) that the present invention synthesizes using sodium periodate method and bovine serum albumin (BSA) is as the coating antigen of colloidal gold strip p-wire (T line), using Salmonella core polysaccharide monoclonal antibody SQX6D8 as gold labeling antibody.The present invention is different from the principle of conventional pathogenic bacterium colloidal gold strip sandwich assay, this Salmonella colloidal gold strip method uses indirect competition principle to detect, and Salmonella core polysaccharide monoclonal antibody specific ensure that Salmonella in belonging to all is had intersection simultaneously to belonging to outer antibacterial no cross reaction by method, and the detection comprehensive, easy, quick belonged to for Salmonella in Food provides analysis means.
Description
Technical field
The present invention relates to Salmonella in a kind of detection food based on Salmonella core polysaccharide monoclonal antibody SQX6D8
The preparation method of the colloidal gold strip of Pseudomonas, belongs to immunoassay field.
Background technology
Salmonella (Salmonella) is a kind of global food-borne pathogens.Biologically Salmonella is a class
The gram negative bacteria of the blunt circle in two ends, without spore, general without pod membrane, major antigen has O antigen, H antigen, Vi antigen.Animality
Food such as Fowl meat, eggs, milk easily pollute Salmonella.Human body is taken in containing causing acute gastroenteritis after bacterium food, typhoid fever,
The crowds such as the child of hypoimmunity even occur the symptoms such as septicemia.
Salmonella has 2000 various serotypes, and serotype common in clinic is mainly Salmonella enteritidis, mouse typhus
Salmonella, Salmonella paratyphi A etc..The production operation process of good specification and hazard contro1
Etc. (HACCP) application of management system can largely reduce the generation of food-borne pathogens.But to raw material and production
Process, the quality-monitoring of product are also the important means ensureing biological food safety.
The method of detection Salmonella mainly has biochemical culture method, immunological detection method, molecular detecting method at present.Pass
The biochemical culture method of system is the national standard method of detection Salmonella, although authority is reliable, but it is generally required to 5-10 days obtain result,
And operating process is loaded down with trivial details, it is impossible to adapt to the requirement of quickly detection；Molecular detecting method is based on salmonella dna
(DNA) polymerase chain reaction (PCR) sets up.Develop at present normal PCR, real-time fluorescence quantitative PCR (RT-PCR),
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP).Compared with normal PCR,
RT-PCR has and realizes detection by quantitative target dna, specificity solution higher, effective PCR pollution problem, the high spy of automaticity
Point.LAMP method has simple, quick, the feature of high specificity.This technology is at aspects such as sensitivity, specificity and detection ranges
No less than Standard PCR technology, it is independent of special instrument and equipment, can quickly detect by on-the-spot high flux, and testing cost is remote
Less than real-time fluorescence quantitative PCR.But, when having document report LAMP method Salmonella in detecting Lac Bovis seu Bubali, it may appear that false
Negative problem.Caused by this is probably and is affected by sample substrate with primer.Same Standard PCR and real-time fluorescence PCR are also
It is faced with the problem that testing cost is high, higher to the requirement of operator's technology.
Colloidal gold strip method detection pathogenic microorganism has simplicity, quick, the advantage of low cost.At present at Pseudomonas water
The colloidal gold strip of flat upper detection Salmonella has no that report, Major Difficulties concentrate on the Dan Ke that intersection is homogeneous, affinity is high
The preparation of grand antibody, and the acquisition of the pairing antibody for double-antibody sandwich.Intersection is we disclosed in patent before
The immunogenic synthesis of property (number of patent application: 201410314040.3), the foundation of chiasma type DAS-ELISA method
(201510183851.9).But when setting up colloidal gold strip method with pairing antibody, it is faced with the problem that T line does not develops the color, its
Middle reason may have: the affinity of the monoclonal antibody that (1) screens meets ELISA but is insufficient for colloidal gold strip and wants
Ask；(2) core polysaccharide is difficult to be exposed to phage surface, in the situation of colloidal gold strip method response time the shortest (10 min)
Under, it is fixed on the Salmonella that the antibody of T line is difficult to effectively capture in sample.Therefore, we use freshly prepd, have higher
Monoclonal antibody SQX6D8 of affinity, and synthesize allos LPS conjugate as T line coating antigen, examine by competition law principle
Survey, overcome antibody on T line and defy capture the difficult problem of Salmonella.The method is sensitive to the detection of 12 strain Salmonellas of test
Degree is 105 - 5×106CFU/mL, intersects the most homogeneous, simultaneously for other test bacterium such as E.coli, E.coli O157:
H7, Enterobacter sakazakii, campylobacter jejuni, campylobacter coli, vibrio parahaemolyticus, staphylococcus aureus, single increasing Liszt
Bacterium no cross reaction.
Summary of the invention
It is an object of the invention to set up a kind of colloidal gold strip detecting Salmonella in Pseudomonas level, be used for eating
In product, the high specific of Salmonella, high accuracy quickly detect.
Technical scheme, for achieving the above object, the present invention establishes a kind of based on Salmonella core polysaccharide
The colloidal gold strip method that the competition law principle detection Salmonella in Food of monoclonal antibody belongs to, the method also includes optimizing
Process.
Wherein, monoclonal antibody SQX6D8 is to use EDC method LPS-BSA artificial antigen as immunogen immune 8 week old
BALB/c mouse also obtains through hybridoma technology fusion, screening, has the advantages that affinity is high, inhibition is good.
Wherein, T line coating antigen uses NaIO4The LPS-BSA conjugate of method synthesis, EDC method LPS-BSA comparing homology presses down
Effect processed is more preferable.
The detection analysis principle of the inventive method: using indirect competition principle to detect, p-wire (T line) is sprayed with synthesis
LPS-BSA conjugate do coating antigen, Salmonella core polysaccharide monoclonal antibody specific SQX6D8 and red gold nano
Particle carries out coupling as gold labeling antibody.During detection, the Salmonella in sample is first combined with gold labeling antibody thus inhibits gold
Labeling antibody and the combination of coating antigen on T line, the colored intensity of T line is inversely proportional to the quantity of Salmonella in sample.Salmonella
Belong to core polysaccharide monoclonal antibody specific and ensure that Salmonella in belonging to all is had intersection simultaneously to belonging to outer antibacterial without handing over by method
Fork reaction.
One strain monoclonal cell strain SQX6D8 based on Salmonella core polysaccharide, has been preserved in Chinese microorganism strain and has protected
Hiding administration committee's common micro-organisms center, be called for short CGMCC, deposit number is CGMCC No.12019.
Monoclonal antibody SQX6D8 based on Salmonella core polysaccharide, it is CGMCC by described deposit number
The monoclonal cell strain SQX6D8 secretion of No.12019 produces.
The colloid that a kind of detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody SQX6D8 belongs to
The preparation method of gold test paper strip, concretely comprises the following steps:
(1) preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8: using deposit number is CGMCC No.12019's
Bacterial strain immunity obtains monoclonal antibody；
(2) synthesis of T line coating antigen LPS-BSA conjugate:
A, activation: take saltant type Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg ultra-pure water and dissolve according to reaction mass ratio
NaIO4Ra-LPS 51 dropping 47 μ L concentration are the NaIO of 200mM/L4Solution is to saltant type Salmonella typhimurium lipopolysaccharide
In Ra-LPS solution, 25 DEG C of reaction 2h；Then the ethylene glycol solution of 10 μ L 1M/L is taken in reactant liquor, 25 DEG C of reaction 2h；
B, coupling: the saltant type mouse typhus sramana after step a being activated according to the ratio that reaction mol ratio Ra-LPS BSA is 51
Salmonella lipopolysaccharide Ra-LPS reactant liquor is added drop-wise in BSA solution, regulates reactant liquor with the carbonate buffer solution CB of 0.01M, pH9.6
PH to 8.5；Room temperature reaction overnight, i.e. obtains the LPS-BSA conjugate of synthesis after dialysis；
(3) synthesis of golden nanometer particle: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
(4) coupling of gold labeling antibody: 1mL colloidal gold solution pH is adjusted to 8 with 0.1M solution of potassium carbonate；Add the Salmonella of 10 μ g
Sclerotium heart Monoclonal Antibody against Polysaccharides SQX6D8 also at room temperature reacts 2h；The BSA solution adding 50 μ L mass volume ratios 10% enters
Row is closed, room temperature reaction 2h；4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove gold colloidal and the list of non-coupling
Clonal antibody；Gold labeling antibody is washed with the phosphate buffer containing 0.2% Tween, the 0.01M of 0.2% sucrose；
(5) preparation of gold label test strip: nitrocellulose filter is fixed on PVC base plate, adsorptive pads is sticked to cellulose nitrate
Sample pad near one end of nature controlling line, is sticked to the close p-wire of nitrocellulose filter and PVC base plate with PVC base plate by element film
The other end；6mm it is spaced between nature controlling line and p-wire；LPS-BSA conjugate Membrane jetter is sprayed onto at p-wire, 0.5mg/
The sheep anti-mouse igg two of mL is anti-to be sprayed onto at nature controlling line；37 DEG C of drying 2h cutting are standby, obtain based on Salmonella core polysaccharide list
The colloidal gold strip that the detection Salmonella in Food of clonal antibody SQX6D8 belongs to.
Described Salmonella includes 12 strains, is followed successively by paratyphoid A (A group), that sramana of Argonne (B group), mouse typhus sramana
(B group), paratyphoid B (B group), Thompson sramana (C1 group), Bu Luokeli sramana (C2 group), Kentucky sramana (C3 group), intestinal
Scorching sramana (D group), typhoid fever sramana (D group), Dublin sramana (D group), duck sramana (E group), Arizona sramana.
Beneficial effects of the present invention: the Salmonella specificity colloidal gold strip that the present invention provides is different from conventional big
The principle of molecular colloid gold test paper strip double antibody sandwich method, but have employed competition law and detect.
Salmonella core polysaccharide specific monoclonal antibody SQX6D8 affinity is higher, it is more uniform to intersect, by closing
The LPS-BSA conjugate become is as T line coating antigen and the gold mark of Salmonella competition SQX6D8 coupling golden nanometer particle in sample
Antibody, the colloidal gold strip of foundation can detect Salmonella in Pseudomonas level, and not intersect instead with other test bacterium
Should.The method is easy and simple to handle quickly, the feature of good stability, low cost, has promotion and application and is worth.
Biological material specimens preservation: monoclonal cell strain SQX6D8, has been preserved in Chinese microorganism strain preservation management and has entrusted
Member's meeting common micro-organisms center, is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro-life of the Chinese Academy of Sciences
Thing institute, preservation date: on January 20th, 2016, deposit number CGMCC No.12019.
Accompanying drawing explanation
The principle schematic of Fig. 1 Salmonella specificity colloidal gold strip.
Fig. 2 Salmonella specificity colloidal gold strip detection saltant type lipopolysaccharide (RaLPS).
Fig. 3 Salmonella specificity colloidal gold strip detection Salmonella.
Fig. 4 Salmonella specificity colloidal gold strip cross reaction.
Detailed description of the invention
Embodiment 1
Concretely comprising the following steps of this colloidal gold strip exploitation:
(1) preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8:
Use number of patent application: the immunogen synthesis method disclosed in 201410314040.3, with 1-(3-dimethylamino-propyl)-
3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) method synthesis saltant type Salmonella lipopolysaccharide
(Ra-LPS) with keyhole limpet hemocyanin (KLH) complete antigen, and as immunogen immune mice, merged by regular growth and
LPS, the Salmonella thalline of different O antigen carry out positive cell screening as coating antigen, are finally prepared for Salmonella
Interior affinity is high, intersects more uniform genus monoclonal antibody specific SQX6D8.
(2) synthesis of T line coating antigen LPS-BSA conjugate: use number of patent application: disclosed in 2014103140403
Synthetic method synthesis of coupling thing, specifically comprises the following steps that
A, activation: take saltant type Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg ultra-pure water and dissolve, according to reaction mass ratio
NaIO4Ra-LPS 51 dropping 47 μ L concentration are the NaIO of 200mM/L4Solution is to saltant type Salmonella typhimurium lipopolysaccharide
In Ra-LPS solution, 25 DEG C of reaction 2h；Then the ethylene glycol solution of 10 μ L 1M/L is taken in reactant liquor, 25 DEG C of reaction 2h；
B, coupling: according to the ratio that reaction mol ratio Ra-LPS BSA is 51 by the saltant type Salmonella typhimurium after activation
Lipopolysaccharide Ra-LPS reactant liquor is added drop-wise in BSA solution, regulates reactant liquor pH extremely with the carbonate buffer solution CB of 0.01M, pH9.6
8.5；Room temperature reaction overnight, i.e. obtains the LPS-BSA conjugate of synthesis after dialysis；
(3) synthesis of golden nanometer particle: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
(4) coupling of gold labeling antibody: 1mL colloidal gold solution pH is adjusted to 8 with 0.1M solution of potassium carbonate；Add the sramana of 10 μ g
Salmonella core polysaccharide monoclonal antibody SQX6D8 also at room temperature reacts 2h；The BSA solution adding 50 μ L 10% (M/V) is carried out
Close, room temperature reaction 2h.4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove gold colloidal and the Dan Ke of non-coupling
Grand antibody；Gold labeling antibody is washed with the phosphate buffer containing 0.2% Tween, the 0.01M of 0.2% sucrose；
(5) preparation of gold label test strip: press the fixing water suction of shown position on polyvinylchloride base plate 6 and nitrocellulose filter 2
Pad 1 and sample pad 5, the good p-wire 4 of labelling and the position of nature controlling line 3 successively, it is spaced 6mm；By LPS-BSA conjugate and sheep anti mouse
IgG bis-anti-(0.5mg/mL) is sprayed onto at p-wire 4 and nature controlling line 3 respectively with Membrane jetter BioJet Quanti3000；37 DEG C of bakings
Dry 2h cutting are standby.
Embodiment 2 use this colloidal gold strip detect Salmonella Ra LPS:
First by Ra LPS(1mg/mL) with the phosphate buffer gradient dilution of 0.01M to 100 ng/mL, 50 ng/mL,
25 ng/mL, 10 ng/mL and 5 ng/mL, the phosphate buffer of blank 0.01M compares；
Use wet method detection Ra LPS subsequently, take golden labeling antibody and the 47 μ L re-suspension liquid of preparation in 7 μ L embodiment 1 step (4)
(0.1% Tween, 0.2% sucrose, the phosphate buffer of the 0.01M of 1% BSA), in microwell plate, mixes with liquid-transfering gun.Will
The Ra LPS of variable concentrations respectively takes 150 μ L and is separately added in different microwell plates, uses liquid-transfering gun mix homogeneously, room temperature reaction 5
Minute；Being inserted in microwell plate by the colloidal gold strip of preparation, room temperature reaction carried out interpretation after 10 minutes.Interpretation foundation: blank
Sample, nature controlling line and p-wire have color simultaneously, and p-wire colour developing is relatively deep, positive.Nature controlling line has color, and p-wire shows
Color is the most shallow or do not develop the color compared with blank sample, negative sample.Concrete testing result is as shown in Figure 2.
Embodiment 3 uses this colloidal gold strip to detect 12 strain Salmonellas:
12 strain bacterial strains are followed successively by paratyphoid A (A group) CMCC 50093, Argonne that sramana (B group) CICC 21586, B-mode pair
Typhoid fever (B group) CMCC 50094, mouse typhus sramana (B group) ATCC 13311, Thompson sramana (C1 group) CICC21480, Bu Luo
Gram sharp sramana (C2 group) CICC 21489, Kentucky sramana (C3 group) CICC 21488, enteritis sramana (D group) ATCC13076, wound
Cold Salmonella (D group) CMCC 50071, Dublin sramana (D group) CICC 21497, duck sramana (E group) CICC21498, Ya Li
That sramana ATCC 13314 of Mulberry.
Concrete detection process is: by the phosphate buffer gradient dilution of the Salmonella pure culture 0.01M of test
To 107CFU/mL, 106CFU/Ml, 105CFU/mL and 104CFU/mL, does blank with diluent, and other process is same
Embodiment 2.Concrete testing result is as shown in Figure 3.
Embodiment 4 tests the cross reaction of Salmonella specificity colloidal gold strip:
8 strains test other antibacterial respectively: staphylococcus aureus, Listeria monocytogenes, Escherichia coli O 157, common large intestine
Bacillus, Cronobacter enterobacteria, vibrio parahaemolyticus, campylobacter jejuni, campylobacter coli.Test concentrations is 5 × 108
CFU/mL, detection process is with embodiment 2, and result is as shown in Figure 4.
Claims (4)
1. strain monoclonal cell strain SQX6D8 based on Salmonella core polysaccharide, has been preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, is called for short CGMCC, and deposit number is CGMCC No.12019.
2. monoclonal antibody SQX6D8 based on Salmonella core polysaccharide, it is characterised in that: it by described deposit number is
The monoclonal cell strain SQX6D8 secretion of CGMCC No.12019 produces.
3. the gold colloidal that a detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody SQX6D8 belongs to
The preparation method of test strips, it is characterised in that concretely comprise the following steps:
(1) preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8: using deposit number is CGMCC No.12019's
Bacterial strain immunity obtains monoclonal antibody；
(2) synthesis of T line coating antigen LPS-BSA conjugate:
A, activation: take saltant type Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg ultra-pure water and dissolve, according to reaction mass ratio
NaIO4Ra-LPS 51 dropping 47 μ L concentration are the NaIO of 200mM/L4Solution is to saltant type Salmonella typhimurium lipopolysaccharide
In Ra-LPS solution, 25 DEG C of reaction 2h；Then the ethylene glycol solution of 10 μ L 1M/L is taken in reactant liquor, 25 DEG C of reaction 2h；
B, coupling: the saltant type mouse typhus sramana after step a being activated according to the ratio that reaction mol ratio Ra-LPS BSA is 51
Salmonella lipopolysaccharide Ra-LPS reactant liquor is added drop-wise in BSA solution, regulates reactant liquor with the carbonate buffer solution CB of 0.01M, pH9.6
PH to 8.5；Room temperature reaction overnight, i.e. obtains the LPS-BSA conjugate of synthesis after dialysis；
(3) synthesis of golden nanometer particle: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
(4) coupling of gold labeling antibody: 1mL colloidal gold solution pH is adjusted to 8 with 0.1M solution of potassium carbonate；Add the Salmonella of 10 μ g
Sclerotium heart Monoclonal Antibody against Polysaccharides SQX6D8 also at room temperature reacts 2h；The BSA solution adding 50 μ L mass volume ratios 10% enters
Row is closed, room temperature reaction 2h；4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove gold colloidal and the list of non-coupling
Clonal antibody；Gold labeling antibody is washed with the phosphate buffer containing 0.2% Tween, the 0.01M of 0.2% sucrose；
(5) preparation of gold label test strip: be fixed on PVC base plate on (6) by nitrocellulose filter (2), adheres to adsorptive pads (1)
At nitrocellulose filter (2) with PVC base plate (6) near one end of nature controlling line (3), sample pad (5) is sticked to celluloid
The other end of the close p-wire (4) of film (2) and PVC base plate (6)；6mm it is spaced between nature controlling line (3) and p-wire (4)；Will
LPS-BSA conjugate Membrane jetter is sprayed onto p-wire (4) place, and the sheep anti-mouse igg two of 0.5mg/mL is anti-is sprayed onto nature controlling line (3) place；
37 DEG C of drying 2h cutting are standby, obtain sramana in detection food based on Salmonella core polysaccharide monoclonal antibody SQX6D8
The colloidal gold strip of Bordetella.
Salmonella in detection food based on Salmonella core polysaccharide monoclonal antibody SQX6D8 the most according to claim 3
The preparation method of the colloidal gold strip of Pseudomonas, it is characterised in that: described Salmonella includes 12 strains, is followed successively by paratyphoid A
(A group), that sramana of Argonne (B group), mouse typhus sramana (B group), paratyphoid B (B group), Thompson sramana (C1 group), Bu Luoke
Profit sramana (C2 group), Kentucky sramana (C3 group), enteritis sramana (D group), typhoid fever sramana (D group), Dublin sramana (D group), duck
Sramana (E group), Arizona sramana.
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| PCT/CN2017/086561 WO2017211214A1 (en) | 2016-06-07 | 2017-05-31 | Preparation method of salmonella-detecting colloidal gold test strip for food based on salmonella core polysaccharide monoclonal antibody |
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| CN110470832B (en) * | 2019-07-30 | 2023-11-14 | 山东第一医科大学(山东省医学科学院) | Hollow bimetallic test strip for simultaneously detecting IL-6, IL-4 and TNF-alpha and preparation method thereof |
| CN111876438A (en) * | 2020-07-07 | 2020-11-03 | 杭州傲锐生物医药科技有限公司 | Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof |
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