CN108008127A - The preparation method and application of Enterobacter sakazakii colloidal gold strip - Google Patents
The preparation method and application of Enterobacter sakazakii colloidal gold strip Download PDFInfo
- Publication number
- CN108008127A CN108008127A CN201711210745.0A CN201711210745A CN108008127A CN 108008127 A CN108008127 A CN 108008127A CN 201711210745 A CN201711210745 A CN 201711210745A CN 108008127 A CN108008127 A CN 108008127A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- preparation
- enterobacter sakazakii
- sakazakii
- antigen
- 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
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The preparation method and application of Enterobacter sakazakii colloidal gold strip, is related to a kind of preparation method and application of colloidal gold strip.It is to solve existing Enterobacter sakazakii detection method detection cycle length, and the problem of testing result is unstable.Method：First, the preparation of Enterobacter sakazakii antigen；2nd, sero-fast preparation；3rd, sero-fast purifying；4th, the specific detection of polyclonal antibody；5th, polyclonal antibody is secondarily purified；6th, the preparation of colloidal gold and the mark of Enterobacter sakazakii polyclonal antibody；7th, the assembling of colloidal gold strip.Application of the Enterobacter sakazakii colloidal gold strip in powdered milk sample detection.The specificity of test strips of the present invention is good, and the detection of test strips is limited to 105CFU/mL.Testing result is stablized, and can quickly obtain testing result.The present invention is used to prepare Enterobacter sakazakii colloidal gold strip.
Description
Technical field
The present invention relates to a kind of preparation method and application of colloidal gold strip.
Background technology
Enterobacter sakazakii (C.sakazakii) is that one kind is widely present in nature, can trigger newborn baby after infection
Film inflammation, septicemia, bacteremia, the gramnegative bacterium of necrotizing enterocolitis and nervous system sequelae.It is bred
Ability and very strong to the resistance of environmental factor, the death rate is up to 20%-50% after infections in infants.C.sakazakii's is fast
Speed detection is of great significance control Enterobacter sakazakii harm.
Traditional detection method not only complex steps, detection cycle length, but also testing result is unstable, it is impossible to really plays pair
The monitoring and control effect of pathogenic bacteria.Molecular biology for detection is easily had a great influence by reagent and environment, and testing cost is opposite
It is expensive；And there is still a need for Zengjing Granule, the advantage compared with immunological detection method in no speed in detection process.
The content of the invention
The present invention is to solve existing Enterobacter sakazakii detection method detection cycle length, and testing result is unstable asks
Topic, there is provided the preparation method and application of Enterobacter sakazakii polyclonal antibody.
The preparation method of Enterobacter sakazakii colloidal gold strip of the present invention, comprises the following steps：
First, the preparation of Enterobacter sakazakii antigen；
2nd, sero-fast preparation；
3rd, the antiserum of preparation is purified by octanoic acid-ammonium sulfate method；
4th, the specific detection of polyclonal antibody；
5th, polyclonal antibody is secondarily purified；
6th, the preparation of colloidal gold and the mark of Enterobacter sakazakii polyclonal antibody
7th, the assembling of colloidal gold strip
Further, Enterobacter sakazakii antigen is Enterobacter sakazakii somatic antigen in step 1.
The preparation method of Enterobacter sakazakii somatic antigen is：
Enterobacter sakazakii activation is inoculated in LB solid mediums, 37 DEG C of constant temperature incubation 24h, picking single bacterium colony is inoculated into
5mL LB fluid nutrient mediums, are seeded to 3L's in 37 DEG C, 180r/min shaking table culture 12h, then by 1% inoculum concentration expansion culture
In LB fluid nutrient mediums, 37 DEG C, 180r/min shaking table culture 24h, obtain bacterial culture fluid；Wherein described Enterobacter sakazakii is purchase
It can buy, strain number is ATCC 29544.
5000r/min centrifuges bacterial culture fluid 10min, collects thalline, is washed with 0.01mol/L pH 7.4PBS buffer solutions
After washing 3 times, the 0.01mol/L pH 7.4PBS buffer solutions precipitated with 5mL are resuspended, add 15 μ L formalin solutions, room
Temperature is lower to place 18h, and then in 4 DEG C, 5000r/min centrifugation 10min, 0.01mol/L pH 7.4PBS buffer solutions are used by precipitation
After washing 3 times, it is resuspended with 0.01mol/L pH 7.4PBS buffer solutions, -20 DEG C save backup.
Further, the secondarily purified polyclonal antibody of miscellaneous bacteria antigen reverse adsorption method is utilized in step 5.
Further, in step 5 colloid gold particle is prepared with trisodium citrate reduction gold chloride method.Wherein colloidal gold
The average diameter of grain is 30nm.
The application of Enterobacter sakazakii colloidal gold strip prepared by the above method in powdered milk sample detection.
Beneficial effects of the present invention：
The present invention prepares anti-C.sakazakii polyclonal antibodies by immune cleaning grade White Rabbit.To obtained antibody
Row purifying, and C.sakazakii colloidal gold immunochromatographydetection detection test paper bars are developed by the form of colloidal gold labeled monoclonal antibody, and it is right
Its progress specificity, sensitivity, shelf-life are evaluated, to provide foundation for the quick detection of C.sakazakii.
Purified by the obtained antiserum of caprylic acid-ammonium, obtain purer antibody.Through antigen reverse adsorption
Afterwards, cross reaction between polyclonal antibody and S.typhimurium is effectively eliminated, prepared height exempts from rabbit-anti C.sakazakii
Polyclonal antibody purity is higher, and specificity is good.
Trisodium citrate reduction method is selected to prepare colloidal gold.The colloidal gold solution of a diameter of 30nm is prepared, the solution is clear
Clearly, it is transparent, take on a red color, be adapted to Enterobacter sakazakii antibody mark.
Present invention adjustment trisodium citrate concentration, prepares 30nm colloid gold particles, beneficial to Enterobacter sakazakii polyclonal antibody
Mark, can the exposed multiple epitopes of Enterobacter sakazakii polyclonal antibody, response is tested Enterobacter sakazakii, sensitivity more preferably, after mark
Sample be not easy to precipitate, antibody concentration is uniform, fast, sensitive with antigen binding speed.
By C.sakazakii, S.aureus, S.typhimurium, S.flexneri, L.monocytogenes,
The dilution of several plants of bacterium of E.coli carrys out test strip specificity, show that prepared test strips are positive with C.sakazakii
Reaction, negative with other several plants of bacterium, specificity is good.The detection of the test strips of preparation is limited to 105CFU/mL.Detection
As a result stablize, can quickly obtain testing result.
The shelf-life of colloidal gold strip is determined by accelerated test, 1d is preserved at 37 DEG C according to test strips, equivalent to
Room temperature preserves one month, judges that the test strips room temperature shelf-life is about 10 months.
Brief description of the drawings
Raw more anti-immunity response curves are immunized for somatic antigen in Fig. 1；
Fig. 2 is that raw more anti-immunity response curves are immunized in broken antigen；
Fig. 3 is immunized for somatic antigen and produces polyvalent antibody potency curve；
Fig. 4 is that generation polyvalent antibody potency curve is immunized in broken antigen；
Fig. 5 is C.sakazakii polyclonal antibody SDS-PAGE electrophoresis after purification；
Fig. 6 analyzes polyclonal antibody specificity for octanoic acid-ammonium sulfate purified antibodies through spot hybridization；
Fig. 7 is that the polyclonal antibody after the processing of antigen reverse adsorption method is special through spot hybridization analysis polyclonal antibody
Property；
Fig. 8 reduces gold chloride method for trisodium citrate and prepares colloidal gold solution；
Fig. 9 is colloidal gold solution UV scanning figure；
Figure 10 is colloidal gold transmission electron microscope picture；
Figure 11 is verification gold labeling antibody stability of solution；
Figure 12 is colloidal gold immuno-chromatography test paper strip structure diagram；
Figure 13 is Enterobacter sakazakii test strips specific test；
Figure 14 tests for Enterobacter sakazakii test strips detection limit；
Figure 15 is Enterobacter sakazakii ELISA test strip food analog sample；
Figure 16 accelerates preservation experiment to determine the test strips shelf-life for 37 DEG C.
Embodiment
Elaborate below to the embodiment of the present invention, following embodiments under premised on technical solution of the present invention into
Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities
Apply example.
Experiment one, prepare rabbit-anti C.sakazakii polyclonal antibodies
Select the full bacterial immunities of C.sakazakii former and immune cleaning grade is big respectively for C.sakazakii bacterial cell disruptions immunogene
White rabbit prepares rabbit-anti C.sakazakii polyclonal antibodies.Prepared antiserum is purified by caprylic acid-ammonium, and is passed through
SDS-PAGE electrophoresis analyzes purified result.Pass through polyclonal antibody potency prepared by ELISA method detection.Pass through spot
Polyclonal antibody specificity prepared by hybrid method and the detection of miscellaneous bacteria antigen reverse adsorption method.
(1) prepared by C.sakazakii immunogenes
C.sakazakii activation is inoculated in LB agar mediums, 37 DEG C of constant temperature incubation 24h, picking single bacterium colony is inoculated into
5mL LB fluid nutrient mediums, are expanded by 1% inoculative proportion and cultivate the LB liquid for being seeded to 3L by 37 DEG C, 180r/min shaking table culture 12h
In body culture medium, 37 DEG C, 180r/min shaking table cultures 24h.Wherein C.sakazakii is is commercially available, strain number ATCC
29544。
It is prepared by somatic antigen (Whole cell antigen, WC Ag)：1mL C.sakazakii nutrient solutions are taken, are applied to
In LB agar plates, plate count is carried out.The remaining bacterial culture fluid 10min of 5000r/min centrifugations, collects thalline.With
After 0.01mol/L pH 7.4PBS buffer solutions wash 3 times, the 0.01mol/L pH 7.4PBS buffer solutions with 5mL will be precipitated
It is resuspended, adds 15 μ L formalin solutions, place 18h at room temperature.4 DEG C, 5000r/min centrifugation 10min, precipitation is used
After 0.01mol/L pH 7.4PBS buffer solutions wash 3 times, it is resuspended with 0.01mol/L pH 7.4PBS buffer solutions, -20 DEG C
Save backup.
It is prepared by bacterial cell disruption cellular antigens (Cell debris antigen, CD Ag)：By C.sakazakii nutrient solutions
5000r/min centrifuges 10min, collects thalline.After precipitation is washed 3 times with 0.01mol/L pH 7.4PBS buffer solutions,
5000r/min centrifuges 10min, precipitation 20mL 0.01mol/L pH 7.4PBS buffer solutions is resuspended, Ultrasonic Cell Disruptor surpasses
Sound crushes (working time 3s, intermittent time 6s, work 40 times, 200Hz, net cycle time 6min), 4 DEG C, 5000r/min centrifugations
After 10min, 0.01mol/L pH 7.4PBS buffer solutions washing precipitation 3 times, it is that somatic cells crush antigen to collect precipitation ,-
20 DEG C save backup.
(2) prepared by C.sakazakii polyclonal antibodies
Before head exempts from one week, animal immune vestibule edge venous blood sampling is as negative control sera, then foot-pad immunization injection card
Jie's seedling, 500 μ L/ are only.Immune for the first time, immunogene is mixed with equivalent Freund's complete adjuvant, subcutaneous 4 points of injections after fully emulsified；
Second and third, four times it is immune, immunogene mix with the freund 's incomplete adjuvant of equivalent, fully emulsified subcutaneous 4 injecting immunes afterwards,
Always immune fixing fabric structure is at 1mL/ or so.Immune rear 7d and 14d auricular veins take blood every time, measure potency.It is immune to terminate
Heart extracting blood afterwards, collects antiserum.
1 bacterial cell disruption antigen immunization protocol of table
(3) C.sakazakii polyclonal antibody purifications
The rabbit anti-serum of preparation is purified by octanoic acid-ammonium sulfate method.Caprylic acid can sink under the conditions of meta-acid
Other protein in the serum of shallow lake, make not containing other protein in addition to IgG in supernatant.And ammonium sulfate is a kind of neutral salt, have
Solubility is high and the advantages of being affected by temperature minimum, antibody surface electric charge is largely neutralized after adding ammonium sulfate, so as to reduce egg
White solubility, makes antibody molecule aggregate and precipitate.
1mL antiserums are taken, 4 DEG C, 10000r/min centrifuges 5min, Aspirate supernatant；With 0.06mol/L pH's 5.0
HAc-NaAc buffer solutions dilute 2 times, and pH to 4.5 is adjusted with the HCl of 1mol/L.4 DEG C are placed in be stirred continuously, it is just pungent according to 60 μ L
Caprylic acid is added dropwise in the ratio of acid/1mL serum, and 2h is stood after persistently stirring 0.5h；4 DEG C, 10000r/min centrifugation 30min,
Take supernatant；The PBS of the 0.1mol/L pH7.4 of 1/10 supernatant volume is added into supernatant, with the NaOH of 1mol/L adjust pH to
7.4；It is stirred continuously at 4 DEG C, the ammonium sulfate of isometric pH7.4 is added dropwise, 4 DEG C stands overnight.4 DEG C, 10000r/min from
Heart 30min, abandons supernatant, and precipitation 0.1mol/L pH7.4PBS are resuspended, and dialyse desalination 3d at 4 DEG C, and one is replaced every 12h
Secondary dialyzate.Solution in bag filter is drawn after PEG20000 concentrations, is gained antibody.
And ultraviolet spectrophotometry is carried out to gained antibody, measure A280nmValue and A260nmValue, calculates IgG content, calculates
Formula is：
IgG content=1.45 × A280nm- 0.74 × A260nm
(4) SDS- polyacrylamide gel electrophoresises
Large biological molecule especially protein has different electric charge and molecular mass, and SDS-PAGE divides according to albumen is opposite
The difference of protonatomic mass subunit and protein isolate.SDS is broken intramolecular and intermolecular hydrogen bond as denaturant and solubilizing agents,
Make molecule unfolding, destroy protein conformation；Dithiothreitol (DTT) makes disulfide bonds between half Guang residue, egg as strong reductant
White matter is depolymerizated into polypeptide chain, and in polyacrylamide gel electrophoresis, different protein is carried out according to the size of molecular mass
Distribution.
(5) C.sakazakii polyclonal antibodies titration
Polyclonal antibody potency is measured using indirect ELISA method, this is a kind of most-often used and sensitive reliable detection
Method, can determine whether the presence of antibody or antigen and the number of amount by shade.Basic step is as follows：
(1) it is anti-to add 96 hole of polystyrene using C.sakazakii as antigen according to the coating concentration in 100 μ L/ holes for coating
In each aperture for answering plate, place for 37 DEG C and be incubated 1h.
(2) liquid in aperture is toppled in washing, is cleaned repeatedly 3 times with 320 μ L/ holes PBST cleaning solutions, is patted dry polyphenyl as far as possible
96 hole reaction plate of ethene.
(3) it is closed as preventing non-specific binding, adds l00 μ L BSA confining liquids to close the sky on hole wall in each aperture
Gap, 37 DEG C of placement 1h.
(4) liquid in aperture is toppled in washing, is cleaned repeatedly 3 times with 320 μ L/ holes PBST cleaning solutions, is patted dry polyphenyl as far as possible
96 hole reaction plate of ethene.
(5) antiserum is added by the continuous doubling dilutions of PBS of antiserum 0.1mol/L pH7.4, is added according to 100 μ L/ holes
To on coated plate, 37 DEG C are placed incubation 1h.
(6) liquid in aperture is toppled in washing, is cleaned repeatedly 3 times with 320 μ L/ holes PBST cleaning solutions, is patted dry polyphenyl as far as possible
96 hole reaction plate of ethene.
(7) enzyme labeling antibody adds goat anti-rabbit igg-HRP according to 100 μ L/ holes, places for 37 DEG C and is incubated 30min.
(8) liquid in aperture is toppled in washing, is cleaned repeatedly 5 times with 320 μ L/ holes PBST cleaning solutions, is patted dry polyphenyl as far as possible
96 hole reaction plate of ethene.
(9) colour developing adds the tmb substrate solution of Fresh, 37 DEG C of lucifuge 15min according to 100 μ L/ holes.
(10) reaction is terminated, colorimetric adds H according to 50 μ L/ holes2SO4Terminate liquid terminates reaction, its colour changed into yellow；Use microplate reader
Each hole light absorption value at 450nm is measured, the greatest dilution of positive reaction is judged as sample to be tested potency.
(6) C.sakazakii polyclonal antibodies specific assay
Rabbit anti-serum specificity is detected by spot hybridization, concrete operation step is as follows：
(1) by C.sakazakii, Staphylococcus aureus, Salmonella typhimurium,
Shigella flexneri, Escherichia coli, Listeria monocytogenes activation are inoculated in LB culture mediums
In, 37 DEG C of shaken cultivation 24h.5000r centrifugations 10min collects thalline, is washed three times and is resuspended with 0.1mol/L pH 7.4PBS,
Bacterial concentration is adjusted to 108CFU/mL。
Wherein S.aureus, S.typhimurium, S.flexneri, E.coli, L.monocytogenes are purchase
Obtaining, the strain number of S.aureus is ATCC25923, and the strain number of S.typhimurium is ATCC14028,
The strain number of S.flexneri is ATCC29931, and the strain number of E.coli is ATCC25922, L.monocytogenes's
Strain number is ATCC19114.
(2) after pvdf membrane being divided into multiple small lattice with pencil, it is soaked in the plate for fill methanol and activates 30s.Activation
Take out immediately afterwards, soak 15min with 0.1mol/L pH 7.4PBS.
(3) PBS solution is blotted with filter paper, puts each 2 μ L of bacterium solution respectively in ready-portioned small lattice center, dry in the shade at room temperature,
Washed three times with 0.1mol/L pH 7.4PBS.
(4) film is soaked in 5% degreasing milk solution, after 37 DEG C of horizontal shaker closing 1h, discards confining liquid, use
0.1mol/L pH 7.4PBS are washed three times.
(5) 2 μ L rabbit anti-serums are separately added into each small lattice center, after 37 DEG C of horizontal shakers react 1h, use 0.1mol/L
PH 7.4PBS are washed three times.
(6) HRP-IgG that 10ml dilutes 5000 times is added into plate, 37 DEG C of horizontal shakers react 1h, use 0.1mol/L
PH 7.4PBS are washed three times.
(7) pvdf membrane is immersed in the DAB nitrite ions of Fresh and developed the color, be placed in 37 DEG C of lucifuge colour developing 10min, use
0.1mol/L pH 7.4PBS are washed three times, and room temperature air-dries observation result.
(7) miscellaneous bacteria antigen reverse adsorption method purified polyclonal antibodies
A certain amount of miscellaneous bacteria antigen is taken in centrifuge tube, 4 DEG C, 12000r/min centrifugation 5min, abandon supernatant, precipitation is used
0.1mol/L pH 7.4PBS are resuspended, and are uniformly mixed.4 DEG C, 12000r/min centrifugation 5min, and with 0.1mol/L pH 7.4PBS
Clean 3 times repeatedly.Cleaned antigen is resuspended, is transferred in new centrifuge tube, adds rabbit-anti C.sakazakii Anti-TNF-αs
Body, is uniformly mixed, 37 DEG C, and 150r/min is incubated 1h.4 DEG C, 12000r/min centrifugation 10min, discard antigen antibody complex and sink
Form sediment, supernatant is miscellaneous bacteria antigen reverse adsorption method purifying gained antibody, by polyclonal antibody after purification and miscellaneous bacteria again into
Row spot hybridization detects.
Result of the test：
It is clear that this experiment selects C.sakazakii somatic antigens and C.sakazakii bacterial cell disruptions cellular antigens to be immunized respectively
Clean level White Rabbit, antiserum titre is monitored by indirect elisa method, and draws potency curve and immune response curve, such as Fig. 1,2
It is shown.Somatic antigen is immunized to obtain serum titer to be risen with moderate tone always, and bacterial cell disruption cellular antigens are immunized to obtain serum
Potency increasess slowly early period, and obvious since the 4th week to rise, two groups of experiment rabbit anti-serum potency started without obvious at the 7th week
Rise and potency is higher, therefore heart extracting blood, collect serum.
(curve 1 is antiserum in Fig. 3, and curve 2 is blank serum) as shown in Figure 3, the serum that somatic antigen is immunized
Whole potency is about 256000；(curve 1 is antiserum in Fig. 4, and curve 2 is blank serum) as shown in Figure 4, bacterial cell disruption cell resists
The immune obtained serum end potency of original is about 64000.The result shows that the antiserum end potency that somatic antigen is immunized is substantially high
In the antiserum end potency that bacterial cell disruption cellular immunity obtains.
The former immune antiserum prepared of the full bacterial immunities of caprylic acid-ammonium purifying C.sakazakii, SDS- are selected in this experiment
The polyclonal antibody of PAGE analyses after purification, as shown in Figure 5.The result shows that caprylic acid-ammonium after purification, obtains purer bar
Band, achievees the purpose that IgG purification.And IgG unwinds as heavy chain and light chain by mercaptoethanol, molecular mass respectively in 55kd and
25kd or so.
Since the phenyl ring of the phenylalanine in protein molecule, tyrosine and trp residue institute band has UV absorption
Property, and at its 280nm wavelength absorptions peak, the protein content of solution and its absorbance value direct proportionality, so sharp
Quantitative measurment is carried out to protein with ultraviolet absorption method.First pass through the A of the antibody of ultraviolet spectrophotometry after purification280Value and
A260Value, substitutes into formula, it is 2mg/mL to obtain IgG content.
By rabbit-anti C.sakazakii polyclonal antibodies after purification and S.aureus, S.typhimurium,
S.flexneri, L.monocytogenes, E.coli carry out dot hybridization, to detect prepared antibody specificity, such as Fig. 6 institutes
Show.The result shows that prepared rabbit-anti C.sakazakii polyclonal antibodies and S.aureus, S.typhimurium, E.coli,
S.flexneri, L.monocytogenes do not have cross reaction, have slight cross reaction with S.typhimurium.A1 in Fig. 6,
a2:C.sakazakii；b1,b2:E.coli；c1,c2:S.flexneri；d1,d2:L.monocytogenes；e1,e2:
S.aureus。
Using miscellaneous bacteria antigen reverse adsorption method, polyclonal antibody is further purified, by Anti-TNF-α after purification
Body carries out dot hybridization again with S.typhimurium, as shown in Figure 7.The result shows that miscellaneous bacteria antigen reverse adsorption method can have
Effect eliminates cross reaction between polyclonal antibody and S.typhimurium, prepared rabbit-anti C.sakazakii polyclonal antibodies
There is no cross reaction with other bacterial strains.Prepared antibody and S.typhimurium have the cross reaction reason to be probably
S.typhimurium antigenic structures complexity easily has cross reaction with gramnegative bacterium antiserum, and S.typhimurium is
Enteric bacilli, with intestinal flora there are common epitope, common antigenic determinant stimulates body generation antibody and common antigen
Generation association reaction, produces cross reaction.A1-5 in Fig. 7:C.sakazakii；f1-5:S.typhimurium.
Experiment two, establish colloidal gold immunochromatographimethod method
Select trisodium citrate reduction gold chloride method prepare colloidal gold solution, by naked eyes judge, ultraviolet scanning spectrum and
Transmission electron microscope analysis judges colloidal gold quality.Pass through naked eyes judgement and A580Explore more grams of colloid gold label rabbit-anti C.sakazakii
Optimal pH and optimum protein labelled amount during grand antibody.By exploring, most preferably again prepared by solution combination and low temperature supercentrifugation
Stable gold labeling antibody solution.And T lines to colloidal gold immuno-chromatography test paper strip and C lines optimum antibody coating concentration are visited
Rope, prepares for colloidal gold immuno-chromatography test paper strip and lays the foundation.
(1) prepared by colloidal gold
Trisodium citrate reduction method is selected to prepare colloidal gold.99mL is added in the 250mL triangular flasks of bubble perchromic acid washing lotion
ddH2O, magnetic agitation heating.Tiny bubble to be had, which is emerged, adds 1mL 1%HAuCl4Solution.Until liquid level has a large amount of bubbles to emit
When going out, 1% citric acid three sodium solutions of 1.2mL are added thereto immediately, continue magnetic agitation.The color of solution is by pale yellow, grey
Color, Dark grey, blueness, navy blue, claret, red change successively, until being changed into bright red completely, close heating, cooling
To room temperature, 4 DEG C save backup balance.
(2) colloidal gold quality analysis
The success or not that gold-marking immunity is quickly tested depends on colloidal gold quality, and colloid gold particle homogeneity is then golden mark
The immune most important thing quickly tested.If gold grain is in irregular shape, with reference to albumen with regard to unstable easily dissociation, and then lead
Cause the excessive phenomenon of background and false negative result；Experiment stability and again is influenced whether if gold grain diameter range of variation is too big
Renaturation.Colloidal gold poor quality, colloidal gold conjugate cannot be quickly completely from glass fibre in immunity-chromatography test
Dissociation, so as to influence test mass.
This experiment selection cools down bright red colloidal gold solution, is detected by ultraviolet-uisible spectrophotometer in visible ray
Continuous spectrum absorbance in the range of 400-700nm, determines the wavelength at maximum extinction peak.Pass through transmission electron microscope observing colloid gold particle
Shape and size distribution, the uniformity and dispersion degree.According to the diameter of colloid gold particle in transmission electron microscope amplification factor and photo
Estimate colloidal gold average diameter.
(3) prepared by gold labeling antibody
1mL colloidal gold solutions are taken to add 0.2M K in 1.5mL centrifuge tubes2CO33 μ L of solution, stand after fully mixing
30min.The rabbit-anti C.sakazakii polyclonal antibodies prepared by 10 μ g are slowly added to, 30min is stood after fully mixing.Add
The BSA of final concentration 1% is closed, and 30min is stood after fully mixing.4 DEG C, 12000r/min, 30min is centrifuged, abandons supernatant, will
100 μ L colloidal golds of precipitation redissolve liquid and are resuspended.
(4) colloidal gold immunochromatographimethod condition
It is 9.5 to mark pH value；Protein labeling amount：10 μ g antibody are added per 1mL colloidal gold solutions；Centrifugal condition：4 DEG C,
12000r/min, 30min；Redissolve liquid and be combined as Tris-Cl+BSA+ sucrose+trehalose；Select T lines antibody coating concentration for
1mg/mL；The antibody coating concentration for selecting C lines is 1mg/mL.
Result of the test：
Trisodium citrate reduction gold chloride method is selected to prepare colloidal gold, as shown in figure 8, observing by the naked eye prepared glue
Body gold solution transparent and homogeneous, are inside visible by naked eyes impurity, bright-colored, and in shiny red, preliminary judgement is suitable for marking more grams
Grand antibody.
By ultraviolet-uisible spectrophotometer in 400-700nm visible-ranges to colloidal gold labeled monoclonal antibody after institute's shape
Into gold labeling antibody solution carry out spectral scan, as shown in Figure 9 (in Fig. 9 solid line represent colloidal gold spectral scan, dotted line represent gold
Labeling antibody spectral scan), form spectral scan figure and integrally moved towards with trend without obvious poor with colloidal gold solution spectral scan figure
Different and fluctuation, remains single absworption peak, there is maximum absorption band at 530.8nm, and OD values are 0.9196；Have at 449.8nm
Minimal absorption peak, OD values are 0.5103.The result shows that combined between colloidal gold and antibody more stable.
By shape and size distribution, the uniformity and dispersion degree of transmission electron microscope observing colloid gold particle, as shown in Figure 10,
The rounded distribution of colloid gold particle, particle diameter and distribution is uniform, and is averaged according to transmission electron microscope preresearch estimates colloid gold particle
A diameter of 30nm.
Prepared gold labeling antibody effect is best, and the clarification of gold labeling antibody solution is visible by naked eyes particle after redissolution, and precipitates
No wall built-up occurs.By the gold labeling antibody after resuspension, put in colloidal gold pad, detect C.sakazakii bacterium solutions, PBS, LB respectively,
Its colour developing situation is observed, (wherein 1 is Cronobacter sakzakii as shown in figure 11；2 be LB；3 be PBS), No. 1 T line and C
Developing the color in various degree all occurs in line, and No. 2 and No. 3 C lines develop the color, and T lines do not develop the color.Show to redissolve liquid proportional and sealing effect compared with
It is good.
Experiment three, prepare colloid gold immune layer test strips
Colloidal gold immuno-chromatography test paper strip is assembled, and prepared test strips are evaluated.By C.sakazakii,
The dilution detection examination of several plants of bacterium of S.aureus, S.typhimurium, S.flexneri, E.coli, L.monocytogenes
The specificity of paper slip.By by the detection limit of 10 times of doubling dilution test strips of C.sakazakii bacterium solutions.And will be prepared
Test strips be applied to inoculation C.sakazakii, S.aureus, S.typhimurium, S.flexneri, E.coli,
The Infant Formula Enterprises test strip of L.monocytogenes is applied to specificity during food samples.Finally by acceleration
Experiment determines the shelf-life of prepared test strips.
(1) test strips assemble
Colloidal gold immuno-chromatography test paper strip is mainly made of four parts：Sample pad (sample pad), bonding pad
(conjugate pad), nitrocellulose filter (NC membrane), water absorption pad (absorbent pad).Colloid gold immune layer
Test strips structure schematic diagram such as Figure 12 is analysed, wherein 1 is sample pad, 2 be bonding pad, and 3 be detection line, and 4 be nature controlling line, and 5 be nitric acid
Cellulose membrane, 6 be PVC bottom plates.
Wherein bonding pad is connected with the both ends of nitrocellulose filter respectively with water absorption pad, sample pad and bonding pad other end phase
Even.This four part is partially overlapped by each other, and detection line (T) and nature controlling line (C) are coated with nitrocellulose filter, forms complete test paper
Bar system.The swimming forward on chromatography strip under capillary diffusion effect after sample adds sample pad, when swimming to bonding pad,
As contained material to be checked in sample, then the immune response of first step high degree of specificity occurs, the immune complex of formation continues to swim
When moving to wire coating area, the immune response of second step high degree of specificity occurs, the immune complex of formation is trapped within coating
Wire area, red stripes are shown by the colloidal gold of mark, Article 2 line is control line, by remaining free label shape
Into on film, represent and test completion.
(2) colloidal gold immuno-chromatography test paper strip specific detection
By C.sakazakii bacterial strains and S.aureus, S.typhimurium, S.flexneri, L.monocytogenes,
E.coli bacterial strains are cultivated respectively, and bacterial concentration is diluted to 10 with 0.01mol/L PBS buffer solutions8CFU/mL, takes respectively
100 μ L are added in test strips, and result is read after 15min.
(3) colloidal gold immuno-chromatography test paper strip detection limit detects
By C.sakazakii bacterium solutions 10 times of doubling dilutions of 0.01mol/L PBS buffer solutions, being diluted to concentration respectively is
108CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL、104CFU/mL、103CFU/mL、102CFU/mL、
10CFU/mL, takes each 100 μ L of concentration bacterium solution to add in the colloidal gold immuno-chromatography test paper strip prepared respectively, and knot is read after 15min
Fruit.
(4) colloidal gold immuno-chromatography test paper strip is detected applied to food samples
Take the commercially available Infant Formula Enterprises of 1g to be dissolved in 5mL sterile waters, respectively be inoculated with C.sakazakii, S.aureus,
S.typhimurium, S.flexneri, L.monocytogenes, E.coli, increase bacterium by 12h, ultraviolet to be in charge of luminosity score
Its OD is not measured600Value, each bacterial concentration is adjusted with sterile water, then with the colloidal gold immuno-chromatography test paper strip prepared to it
It is detected.
2 simulated foods sample list of table
(5) the colloidal gold immuno-chromatography test paper strip shelf-life determines
This experiment determines the shelf-life of prepared colloidal gold immuno-chromatography test paper strip by accelerated test.According to test strips
1d is preserved at 37 DEG C, preserves equivalent to room temperature and is determined over one month.Test strips are placed in 37 DEG C of preservations, in 7d, 8d,
It is 10 to concentration that 9d, 10d, 11d take test strips respectively4CFU/mL、105CFU/mL、106CFU/mL、107CFU/mL、
108The C.sakazakii dilutions of CFU/mL are detected, by judging the aobvious of colloidal gold immuno-chromatography test paper strip C lines and T lines
Pornographic condition judges the shelf-life of test strips.
Result of the test：
This experiment by respectively by C.sakazakii, S.aureus, S.typhimurium, S.flexneri, E.coli,
L.monocytogenes dilutions are added drop-wise to test strips and carry out test strip specificity, as shown in figure 13.As seen from the figure, detect
C lines develop the color during C.sakazakii, the colour developing of T lines；Detect S.aureus, S.typhimurium, S.flexneri,
The C lines colour developing of test strips, T lines do not develop the color when L.monocytogenes, E.coli.Prepared test strips are in C.sakazakii
Positive reaction, negative with other several plants of bacterium, test strips specificity is good.
This experiment by C.sakazakii bacterium solutions 0.01mol/L PBS solutions by distinguishing 10 times of doubling dilutions extremely
108CFU/mL、107CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL、103CFU/mL、102CFU/mL, 10CFU/mL,
Several groups of dilutions are added drop-wise to prepared test strips respectively, carry out test strip detection limit, as shown in figure 14.
As seen from the figure, prepared ELISA test strip 108CFU/mL、107CFU/mL、106CFU/mL、105C lines during CFU/mL,
T lines have obvious colour developing, are positive.Detection 104CFU/mL、103CFU/mL、102Test strips C when CFU/mL, 10CFU/mL
Line develops the color, and T lines do not develop the color.Therefore, judge that the test strips detection of this experimental study is limited to 105CFU/mL。
This experiment respectively by C.sakazakii, S.aureus, S.typhimurium, S.flexneri,
L.monocytogenes, E.coli are inoculated with commercially available Infant Formula Enterprises, and carry out increasing bacterium, with the colloidal gold immunochromatographimethod of preparation
Test strips are detected, as a result as shown in figure 15.Wherein 1 is C.sakazakii；2 be S.aureus；3 are
S.typhimurium；4 be S.flexneri；5 be L.monocytogenes；6 be E.coli.As seen from the figure, detection inoculation
During the Infant Formula Enterprises of C.sakazakii, test strips C lines and T lines all substantially develop the color, and display is positive；And detect and be inoculated with other
During the Infant Formula Enterprises of bacterium, T lines do not develop the color, and display is negative.Therefore, prepared test strips are detected food samples,
It is specific good.
This experiment preserves 1d by the way that test strips are placed in 37 DEG C of preservations, according to test strips at 37 DEG C, is preserved equivalent to room temperature
One month, the colloidal gold strip shelf-life is determined by accelerated test, as shown in figure 16.Understand test strips being placed in 37 DEG C of guarantors
7d is deposited, T lines and the colour developing of C lines are normal；37 DEG C of preservation 10d are placed in, T lines and C lines colour developing situation have faint decline, but do not influence test paper
Bar quality；37 DEG C of preservation 11d are placed in, T lines and C lines colour developing situation decline, and 106CFU/mL group test strips T lines do not develop the color.Therefore
Judge that the test strips room temperature shelf-life is about 10 months.
Claims (6)
1. the preparation method of Enterobacter sakazakii colloidal gold strip, it is characterised in that this method comprises the following steps：
First, the preparation of Enterobacter sakazakii antigen；
2nd, sero-fast preparation；
3rd, the antiserum of preparation is purified by octanoic acid-ammonium sulfate method；
4th, the specific detection of polyclonal antibody；
5th, polyclonal antibody is secondarily purified；
6th, the preparation of colloidal gold and the mark of Enterobacter sakazakii polyclonal antibody；
7th, the assembling of colloidal gold strip.
2. the preparation method of Enterobacter sakazakii colloidal gold strip according to claim 1, it is characterised in that in step 1
Enterobacter sakazakii antigen is Enterobacter sakazakii somatic antigen.
3. the preparation method of Enterobacter sakazakii colloidal gold strip according to claim 2, it is characterised in that the rugged intestines bar of slope
The preparation method of bacterium somatic antigen is：
Enterobacter sakazakii activation is inoculated in LB solid mediums, 37 DEG C of constant temperature incubation 24h, picking single bacterium colony is inoculated into 5mL LB
Fluid nutrient medium, expands culture and is seeded to the LB liquid of 3L in 37 DEG C, 180r/min shaking table culture 12h, then by 1% inoculum concentration
In culture medium, 37 DEG C, 180r/min shaking table culture 24h, obtain bacterial culture fluid；
5000r/min centrifuges bacterial culture fluid 10min, collects thalline, 3 are washed with 0.01mol/L pH 7.4PBS buffer solutions
After secondary, the 0.01mol/L pH 7.4PBS buffer solutions precipitated with 5mL are resuspended, add 15 μ L formalin solutions, at room temperature
18h is placed, then in 4 DEG C, 5000r/min centrifugation 10min, 3 are washed by precipitation with 0.01mol/L pH 7.4PBS buffer solutions
After secondary, it is resuspended with 0.01mol/L pH 7.4PBS buffer solutions, -20 DEG C save backup.
4. the preparation method of Enterobacter sakazakii colloidal gold strip according to claim 1 or 2, it is characterised in that step 5
It is middle to utilize the secondarily purified polyclonal antibody of miscellaneous bacteria antigen reverse adsorption method.
5. the preparation method of Enterobacter sakazakii colloidal gold strip according to claim 4, it is characterised in that in step 5
Colloid gold particle is prepared with trisodium citrate reduction gold chloride method.Wherein the average diameter of colloid gold particle is 30nm.
6. the application of Enterobacter sakazakii colloidal gold strip prepared by method as claimed in claim 1 in powdered milk sample detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711210745.0A CN108008127B (en) | 2017-11-28 | 2017-11-28 | Preparation method and application of enterobacter sakazakii colloidal gold test strip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711210745.0A CN108008127B (en) | 2017-11-28 | 2017-11-28 | Preparation method and application of enterobacter sakazakii colloidal gold test strip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108008127A true CN108008127A (en) | 2018-05-08 |
| CN108008127B CN108008127B (en) | 2020-09-04 |
Family
ID=62052477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711210745.0A Active CN108008127B (en) | 2017-11-28 | 2017-11-28 | Preparation method and application of enterobacter sakazakii colloidal gold test strip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108008127B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110726847A (en) * | 2019-11-01 | 2020-01-24 | 厦门大学 | Method for detecting estrogen pollution of water body based on recombinant bostrichthys sinensis vitellogenin and application |
| CN112834749A (en) * | 2020-12-14 | 2021-05-25 | 黑龙江大学 | Preparation method and application of test paper for detecting cronobacter sakazakii |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101482563A (en) * | 2009-02-13 | 2009-07-15 | 广东省微生物研究所 | Enterobacter sakazaii enriched reagent and its production method |
| CN102135541A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of enterobacter sakazakii and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| KR20110103579A (en) * | 2010-03-15 | 2011-09-21 | 영남대학교 산학협력단 | A polyclonal antibody of anti-enterobacter sakazakii |
| CN105693855A (en) * | 2014-11-24 | 2016-06-22 | 中国海洋大学 | Method for efficiently preparing Enterobacter sakazakii polyclonal antibody |
-
2017
- 2017-11-28 CN CN201711210745.0A patent/CN108008127B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101482563A (en) * | 2009-02-13 | 2009-07-15 | 广东省微生物研究所 | Enterobacter sakazaii enriched reagent and its production method |
| KR20110103579A (en) * | 2010-03-15 | 2011-09-21 | 영남대학교 산학협력단 | A polyclonal antibody of anti-enterobacter sakazakii |
| CN102135541A (en) * | 2010-09-03 | 2011-07-27 | 李克生 | Detection method of enterobacter sakazakii and gold-labeled rapid diagnosis kit for same and preparation method thereof |
| CN105693855A (en) * | 2014-11-24 | 2016-06-22 | 中国海洋大学 | Method for efficiently preparing Enterobacter sakazakii polyclonal antibody |
Non-Patent Citations (1)
| Title |
|---|
| 黎鹏: "阪崎肠杆菌快速检测方法的建立及其特异性抗原的筛选与验证", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110726847A (en) * | 2019-11-01 | 2020-01-24 | 厦门大学 | Method for detecting estrogen pollution of water body based on recombinant bostrichthys sinensis vitellogenin and application |
| CN112834749A (en) * | 2020-12-14 | 2021-05-25 | 黑龙江大学 | Preparation method and application of test paper for detecting cronobacter sakazakii |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108008127B (en) | 2020-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Seo et al. | Development of a rapid response biosensor for detection of Salmonella typhimurium | |
| Tam et al. | Serological classification of Neisseria gonorrhoeae with monoclonal antibodies | |
| Usleber et al. | Direct enzyme immunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish | |
| Jin et al. | Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products | |
| CN107202884B (en) | Based on immunomagnetic beads and MnO2Nanoparticle detects vibrio parahemolyticus kit | |
| CN104792991B (en) | The specific diabodies sandwich assay that a kind of detection Salmonella in Food based on monoclonal antibody belongs to | |
| CN104316690B (en) | Vibrio parahemolyticus immune colloid gold Rapid detection test strip | |
| CN107045061A (en) | Based on Magnetic Isolation and quantum dot-labeled vibrio parahemolyticus detection kit | |
| Shim et al. | Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test | |
| Peedel et al. | Rapid biosensing of Staphylococcus aureus bacteria in milk | |
| CN105646715B (en) | A kind of monoclonal antibody and its application of anti-fancy carp Immunoglobulin IgM | |
| CN108008127A (en) | The preparation method and application of Enterobacter sakazakii colloidal gold strip | |
| Zhang et al. | Rapid detection of Enterobacter cloacae by immunomagnetic separation and a colloidal gold-based immunochromatographic assay | |
| CN109709319A (en) | A kind of preparation method of salmonella immunomagnetic beads | |
| CN102095855A (en) | Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit | |
| CN109517802A (en) | Secrete hybridoma cell strain, its monoclonal antibody and its application of Salmonella Pullorm IpaJ monoclonal antibody | |
| CN107727853B (en) | Vibrio splindidus colloidal gold immuno-chromatography test paper strip and preparation method | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN114574448A (en) | Hybridoma cell strain secreting ActA monoclonal antibody and application thereof | |
| CN101597334A (en) | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application | |
| CN108588033A (en) | Hybridoma cell strain, CD31 monoclonal antibodies, preparation method and application | |
| CN103421747B (en) | Hybridoma cell strain capable of secreting bovine IL-4 monoclonal antibody, monoclonal antibody secreted by hybridoma cell strain and application of hybridoma cell strain | |
| CN113607949A (en) | Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland | |
| CN105486871B (en) | A kind of quick detection canine parvovirus antibody blood clotting suppresses colloidal gold strip, kit and the detection method of potency | |
| CN103911347A (en) | Monoclonal antibody capable of resisting Listeria monocytogenes, and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230216 Address after: Window No.205, 2F (213, 214/215), Experimental Building, Jilin Northeast Asia Cultural and Creative Science Park, No. 155, Jinhe Street, High-tech Development Zone, Changchun City, Jilin Province, 130000 Patentee after: Jilin Province Hollesi Photoelectric Technology Co.,Ltd. Address before: 150080 No. 74, Xuefu Road, Nangang District, Heilongjiang, Harbin Patentee before: Heilongjiang University |
|
| TR01 | Transfer of patent right |