CN104792991B - The specific diabodies sandwich assay that a kind of detection Salmonella in Food based on monoclonal antibody belongs to - Google Patents
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Abstract
The specific diabodies sandwich assay that a kind of detection Salmonella in Food based on monoclonal antibody belongs to, belongs to immunoassay field.Present invention sodium periodate method by the BALB/c mouse of smooth type Salmonella typhimurium LPS coupling bovine serum albumin immunity 7 week old, through immunity, merges, screens to obtain the LPS monoclonal antibody of 12 strain specific recognition Salmonellas.12 strain antibodies labelling horseradish peroxidase HRP respectively also matches two-by-two with Salmonella paratyphi A thalline.Using 8G3 as coated antibody, 8G3 HRP, as detection antibody, sets up detection Salmonella specific diabodies sandwich assay, and detection Salmonella paratyphi A LOD is 10^6CFU/mL.This method all has cross reaction with the bacterium in Salmonella, and with E.coli, E.coli O157:H7, Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes no cross reaction, for Salmonella in Food belong to detection provide analysis means comprehensive, reliably, rapidly and efficiently.
Description
Technical field
The present invention relates to the double antibody sandwich method that a kind of detection by quantitative Salmonella in Food belongs to, belong to immunoassay neck
Territory.
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.
ELISA becomes the conventional inspection of food-borne pathogens by the feature that it is sensitive, quick, specificity is good, easy to spread
Survey method.The difficulty that ELISA method detection Salmonella faces at present is difficult to preparation exactly and identifies Salmonella in Pseudomonas level
The monoclonal antibody of bacterium, the method specificity hence set up is the strongest, it is impossible to other Salmonella in detection Salmonella.It
The patent application of our laboratory front, application number 201310506513.5, denomination of invention " a kind of detection based on monoclonal antibody
The double antibody sandwich method of Salmonella typhimurium in food ", mix as immunogen with LPS and Salmonella typhimurium thalline,
It is prepared for the specific monoclonal antibody of Salmonella typhimurium and establishes the sandwich method ELISA of detection Salmonella typhimurium
Method, LOD is 500cfu/mL.But, LPS and Salmonella thalline mixed immunity play the mainly thalline LPS's of immunization
O specific side chains (LPS is non-thymus-dependent antigen, individually typically only causes humoral immunization), the core polysaccharide of Salmonella by
Causing effective immunne response in sterically hindered being difficult to, the monoclonal antibody therefore screened and the sandwich ELISA specificity of foundation are relatively strong,
Specificity is for detection Salmonella typhimurium, it is impossible to meet the actual sample needs detection specific need of Salmonella
Ask.
In the face of this difficult point, we pass through sodium periodate method (NaIO4) by weak immunogenic LPS and carrier protein couplet
It is prepared for LPS complete antigen.This complete antigen be thymus-dependent antigen compare LPS immunogenicity strengthen and can be with inducing mouse
Produce IgG antibody-like, it is often more important that the serum of preparation all has cross reaction to the Salmonella in Salmonella.Relatively light
Slip LPS we have found that people prepared by normal light slip LPS after the cross reaction of the mice serum of RaLPS artificial antigen immunity
Work antigen can produce the effect as RaLPS artificial antigen, and this is possibly due to smooth type LPS Yu RaLPS and all contains sand
The reason of door Salmonella core polysaccharide (LPS core structure).Smooth type LPS has cost more in terms of preparation immunogen
Low advantage, therefore, uses LPS to synthesize artificial antigen and as immunogen immune mice and carries out merging screening in the present invention,
It is successfully prepared the monoclonal antibody of specific recognition Salmonella and establishes and in Pseudomonas level, detect the double of Salmonella
Antibody sandwich detects, and provides reliable means for quickly detection Salmonella in Food.
Summary of the invention
(1) to solve the technical problem that
It is an object of the invention to set up a kind of MBP enzyme linked immuno-adsorbent assay detecting Salmonella in Pseudomonas level
Method, for Salmonella in Food belong to batch, quickly detect.
(2) technical scheme
For achieving the above object, the present invention establishes what a kind of detection Salmonella in Food based on monoclonal antibody belonged to
Specific diabodies sandwich assay, the method includes the optimization to detection method.
Wherein, monoclonal antibody be use LPS-BSA artificial antigen as immunogen immune 7 week old BALB/c mouse also
Merge through hybridoma technology, screening obtains.
Wherein, the antibody for pairing is to be matched by sandwich assay under parameters optimization, and is determined by test of many times screening
, there is good stability, highly sensitive feature.
Wherein, the sandwich assay of foundation optimizes the concentration of coated antibody, is coated liquid, confining liquid, standard dilutions, detection
Antibody diluent, detects antibody diluted concentration.
The detection analysis principle of the inventive method is:
Being coated coated antibody 8G3 in ELISA Plate, this antibody can capture the Salmonella in Salmonella by specificity；Wash
Plate 3 times, washes away unconjugated antibody, adds confining liquid 220 μ L and closes unnecessary binding site on plate hole；Wash plate 3 times, add sample
And comparison, hatch 1h for 37 DEG C；Wash plate 3 times, add detection antibody 8G3-HRP, hatch 1h for 37 DEG C；Wash plate 4 times, add nitrite ion and show
Color 10min.If sample has enough Salmonellas, then the coated antibody capture of Salmonella and with detection antibody 8G3-
HRP combines, and catalytic substrate produces absorption value (P/N >=2.1) at 450nm, and is judged as the positive；If sample Salmonella
Concentration the lowest (P/N ＜ 2.1) so sample is not captured or capture the least being not enough to of quantity causes enough signals, is determined
For feminine gender.
Antibody 8G3, i.e. cell strain O, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number: CGMCC No.9804.
Step is:
(1) preparation of Salmonella chiasma type LPS cell strain of monoclonal antibody
With synthesis Salmonella LPS-BSA artificial antigen be immunogen, the BALB/c mouse of immune 8 week old, through immunity,
Merging, screening uses the Salmonella of different sero-group and the cross reaction of Ra LPS test cell strain and selects to have and hand over more by force
The cell of fork reaction carries out sub-clone, has finally given the Salmonella monoclonal antibody specific of 12 strain chiasma types；
(2) the pairing screening of monoclonal antibody
12 strain Salmonella monoclonal antibody specifics distinguish labelling horseradish peroxidase HRP, direct method after purification
Carrying out sandwich assay pairing after identification marking success, pairing parameter is as follows: coated antibody 8G3 4 μ g/mL；Be coated liquid be pH9.6,
The carbonate buffer solution of 0.01M；Mark product concentration 5 × 10^7CFU/mL；The PBS of mark product diluent pH7.2,0.01M；Detection antibody
8G3-HRP dilutes 1000 times of uses, and with this understanding, Success in Experiment has obtained the pairing of 10 couples of P/N > 5；
(3) foundation of Salmonella specific ELISA method
Select the pairing that detection limit is stable, sensitive, establish Salmonella with coated antibody 8G3 and detection antibody 8G3-HRP
Pseudomonas specific sandwich ELISA assay method；Design parameter is as follows:
Coated antibody 8G3 is coated concentration: 4 μ g/mL,
It is coated liquid: pH9.6,0.01M carbonate buffer solution,
Mark product diluent: the Tris-HCl solution of pH7.2,0.01M containing 3.3mM EDTA,
Detection antibody concentration: 2 μ g/mL,
Response time: be coated, close: 37 DEG C, 2h；Standard substance: 37 DEG C, 1h；Detection antibody 37 DEG C, 1h；Colour developing 10min；
This ELISA to the Salmonella paratyphi A (A group) in Salmonella, Salmonella typhimurium (B group), Ah
That Salmonella of tribute (B group), moscow' paratyphi B (B group), Thompson Salmonella (C1 group), Bu Luokeli Salmonella
Bacterium (C2 group), salmonella kentucky (C3 group), Salmonella enteritidis (D group), salmonella dublin (D group), typhoid fever sramana
Salmonella (D group), Salmonella anatis (E group), Arizona Salmonella (Arizona subgenus) all have cross reaction, corresponding inspection
Survey limit (P/N >=2.1) and be respectively as follows: 1.5 × 10^6CFU/mL, 1.5 × 10^6CFU/mL, 6.3 × 10^6CFU/mL, 1.3 × 10^
7CFU/mL、3.1×10^6CFU/mL、3.1×10^6CFU/mL、6.3×10^6CFU/mL、6.3×10^6CFU/mL、2.5×
10^7CFU/mL、2.5×10^7CFU/mL、6.3×10^6CFU/mL、2.5×10^7CFU/mL；With E.coli, E.coli
O157:H7, Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes no cross reaction.
(3) beneficial effect
The Salmonella double-antibody sandwich detection method that the present invention provides is based on Salmonella specific LPS monoclonal
Antibody, the sandwich assay of foundation can detect Salmonella in Pseudomonas level.Simultaneous Stabilization is good, low cost, can detect simultaneously
A large amount of samples, applicable food service industry is extensive, high flux, quick, sensitive testing requirement, has promotion and application and is worth.
Biological material specimens preservation: monoclonal cell strain O, has been preserved in China Committee for Culture Collection of Microorganisms general
Logical microorganism center, cylinder claims CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute, deposit number: CGMCC No.9804, preservation date on October 15th, 2014.
Accompanying drawing explanation
The cross reaction of Fig. 1 Salmonella monoclonal antibody specific 8G3.
The standard curve of Fig. 2 Salmonella specific diabodies sandwich assay.
Detailed description of the invention
Further illustrate the present invention by the following examples.
One, instrument:
TGL-40B table-type low-speed centrifuge, Anting Scientific Instrument Factory, Shanghai
KFLOW water purification machine, Kai Folong company
ZD 9556 horizontal shaker, granary science and education equipment factory
The 96 removable ELISA Plate in hole 8 × 12, Xiamen experiment equipment company limited of happy Jiamei
MuLtiska Mks microplate reader, Thermo Labsystems company
Adjustable pipettor, Thermo Labsystems company
Turbine mixer, Shanghai Hu Xi Instrumental Analysis factory
Two, reagent:
Tetramethyl benzidine (TMB), Shanghai Jingchun Industrial Co., Ltd.
Other reagent are analytical reagent
Three, step
1, the preparation of monoclonal antibody
1) laboratory animal: select the BALB/c mouse of 57 week old to carry out immunity；
2) antigen configuration: with sodium periodate method (NaIO4) that the Salmonella typhimurium LPS(of smooth type is purchased from Sigma is public
Department) carry out coupling with bovine serum albumin (BSA), it is thus achieved that LPS-BSA artificial antigen；Concrete operation method is as follows: take smooth type
Salmonella typhimurium lipopolysaccharide LPS 10mg ultra-pure water dissolves, according to reaction mol ratio NaIO4: LPS=9:1 drips difference
The 200mM/L NaIO of volume4Solution in smooth type Salmonella typhimurium lipopolysaccharide LPS solution, 25 DEG C of reaction 2h；Then
Take the ethylene glycol solution of 10 μ L 1M/L in reactant liquor, 25 DEG C of reaction 2h.Ratio according to reaction mol ratio LPS BSA=5 1
Smooth type Salmonella typhimurium lipopolysaccharide LPS reactant liquor after activation is added drop-wise in BSA solution, with 0.01M, pH9.6
Carbonate buffer solution (CB) regulation reactant liquor pH is to meta-alkalescence.Room temperature reaction overnight, obtains exempting from of Salmonella chiasma type antibody
Epidemic focus；
Using LPS-BSA artificial antigen as immunogen normal saline dilution, it is made into the solution of 2mg/mL；
3) emulsifying: complete to above-mentioned solution and equivalent or incomplete freund adjuvant mix and blend method is carried out emulsifying, emulsifying
Subcutaneous multi-point injection mice after completely；
4) immunity: according to specific immunity flow process immune mouse, 3 exempt from after measure titer with indirect competitive, titer reach want
After asking, carry out spurt immunity；Punching is merged after exempting from posterior orbit blood sampling in 3 days；
5) blood sampling: carry out docking blood sampling for after immunity 1 week for the third time, uses indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure anti-blood
Clear titer；
6) merge, screen: use hybridoma technology to merge, use indirect Elisa to screen positive cell hole, use
Limiting dilution assay carries out sub-clone to positive hole；
7) purification of antibody and preservation: use octanoic acid-saturated ammonium sulfate method purification ascites, obtain monoclonal anti after dialysis
Body, puts into-20 DEG C of preservations after subpackage after using trace UV process to measure its concentration.
2, ELISA course of reaction:
Antibody titer determination step:
1) as serial dilution, coating antigen being coated 96 hole ELISA Plate with being coated buffer, 100 μ L/ holes, in 37 DEG C of incubations
Incubation 2 h in case.Being dried by plate after taking out ELISA Plate, 200 μ L PBST solution are injected in every hole, and shaking table vibrates 3 min, firmly
Get rid of cleaning mixture, absorbent paper pats dry, continue washing 2 times.Following washing methods is identical；
2) after fully washing, with Block buffer sealase target, 200 μ L/ holes, incubation 2 h in 37 DEG C of incubation casees
Rear taking-up is dried stand-by；
3) positive serum serial dilution correspondence is joined 7 ranks before ELISA Plate, eighth row addition negative serum, 100
μ L/ hole, 37 DEG C hatch 1 h after wash, pat dry；
4) every hole adds the sheep anti-mouse igg of HRP labelling of 100 μ L, 1:3000 dilution, 37 DEG C hatch 1 h after wash, clap
Dry；
5) every hole adds 100 μ L nitrite ions (TMB and substrate solution ratio are 1:5), 37 DEG C, dark place reaction, 15 min, takes out
Rear every hole adds 100 μ L stop buffers (sulphuric acid of 2 mol/L), measures light absorption value A by microplate reader450；
Salmonella specific diabodies sandwich method for determining step:
A, it is coated: with the 8G3 coated elisa plate of 4 μ g/mL, 100 μ L/hole, incubation 2 h in 37 DEG C of incubation casees；
B, washing: use PBST washing reaction plate three times, each 3min, 200 μ L/ holes, then dry Sptting plate；
C, closing: containing the CBS of 0.2% gelatin, 200 μ L/hole, close 2h for 37 DEG C；
D, washing: same to b；
E, sample: Salmonella is diluted to 1*10^8,5*10^7,2.5*10^7,1.25 * 10^7,6.25* with PBST
10^6,3.13 * 10^6,1.6 * 10^6 CFU/mL series concentration, separately set a PBST blank.Every hole adds 100 μ L samples
Product, in 37 DEG C of incubation lh；
F, washing: same to b；
G, add detection antibody (8G3-HRP, 2 μ g/mL), 100 μ L/hole, 37 DEG C reaction 1h；
H, washing: same to b；
I, colour developing: add substrate TMB 100 μ L/hole, develop the color 10min；
J, termination: add stop buffer 50 μ L/hole；
K, mensuration: detect OD by microplate reader450nm。
The mensuration of crossing-over rate
By Salmonella paratyphi A (A group), Salmonella typhimurium (B group), that Salmonella of Argonne (B group), second
Type salmonella paratyphi (B group), Thompson Salmonella (C1 group), Bu Luokeli Salmonella (C2 group), Kentucky sramana
Salmonella (C3 group), Salmonella enteritidis (D group), salmonella dublin (D group), salmonella typhi (D group), Salmonella anatis
(E group), Arizona Salmonella (Arizona subgenus) be diluted to 1*10^8,5*10^7,2.5*10^7,1.25 * 10^7,
6.25*10^6,3.13 * 10^6,1.6 * 10^6 CFU/mL series concentration also detect.By E.coli, E.coli O157:H7,
Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes are diluted to 10^8 CFU/mL at the Salmonella double antibody set up
Detecting light absorption value in sandwich assay system, and set blank well, each concentration is done 6 times and is measured meansigma methods, does 3 times and repeats experiment.
Result of the test is as follows:
1, standard curve: the detection range of the Detection of antigen that the present invention is obtained is for 10^6～10^8 CFU/mL, R2=
0.98, specifically ask for an interview Figure of description.
2, the antigen concentration that the standard deviation of the blank absorption value that the mean absorbance that LOD:LOD is blank adds 3 times is corresponding,
Salmonella double antibody sandwich method is 10^6 CFU/mL to the LOD of Salmonella paratyphi A.
3, cross reaction
Result: all colour developings of Salmonella Pseudomonas, to Salmonella paratyphi A (A group), Salmonella typhimurium (B
Group), that Salmonella of Argonne (B group), moscow' paratyphi B (B group), Thompson Salmonella (C1 group), Bu Luokeli
Salmonella (C2 group), salmonella kentucky (C3 group), Salmonella enteritidis (D group), salmonella dublin (D group), wound
Cold Salmonella (D group), Salmonella anatis (E group), Arizona Salmonella (Arizona subgenus).And 10^8 CFU/mL is dense
Other test strain E.coli of degree, E.coli O157:H7, Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes
Do not develop the color (OD < 0.15).Illustrate that the Salmonella sandwich ELISA method set up has Salmonella specificity, with
E.coli, E.coli O157:H7, Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes no cross reaction, specificity
Well.
Claims (1)
1. the specific diabodies sandwich assay that a detection Salmonella in Food based on monoclonal antibody belongs to, it is characterised in that
Being coated coated antibody 8G3 in ELISA Plate, can capture the Salmonella in Salmonella by specificity, coated antibody 8G3 is with husky
After door Salmonella combines, catalytic substrate produces absorption value at 450nm, is judged as the positive during P/N >=2.1, otherwise, P/N ＜ 2.1 is then
It is judged to feminine gender；
Antibody 8G3 is produced by the secretion of monoclonal cell strain O；Cell strain O has been preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, deposit number: CGMCC No.9804；
Step is:
(1) preparation of Salmonella chiasma type LPS cell strain of monoclonal antibody
It is immunogen with the Salmonella LPS-BSA artificial antigen of synthesis, the BALB/c mouse of immune 8 week old, through immunity, melt
Closing, screening uses the Salmonella of different sero-group and the cross reaction of Ra LPS test cell strain and selects to have and intersect more by force
The cell of reaction carries out sub-clone, has finally given the Salmonella monoclonal antibody specific of 12 strain chiasma types；
(2) the pairing screening of monoclonal antibody
12 strain Salmonella monoclonal antibody specifics distinguish labelling horseradish peroxidase HRP after purification, and direct method is identified
Labelling success laggard row sandwich assay pairing, pairing parameter is as follows: coated antibody 8G3 4 μ g/mL；Being coated liquid is pH9.6,0.01M
Carbonate buffer solution；Mark product concentration 5 × 107CFU/mL；The PBS of mark product diluent pH7.2,0.01M；Detection antibody 8G3-
HRP dilutes 1000 times of uses, and with this understanding, Success in Experiment has obtained the pairing of 10 couples of P/N > 5；
(3) foundation of Salmonella specific ELISA method
Select the pairing that detection limit is stable, sensitive, establish Salmonella with coated antibody 8G3 and detection antibody 8G3-HRP
Specific sandwich ELISA assay method；Design parameter is as follows:
Coated antibody 8G3 is coated concentration: 4 μ g/mL,
It is coated liquid: pH9.6,0.01M carbonate buffer solution,
Mark product diluent: the Tris-HCl solution of pH7.2,0.01M containing 3.3mM EDTA,
Detection antibody concentration: 2 μ g/mL,
Response time: be coated, close: 37 DEG C, 2h；Standard substance: 37 DEG C, 1h；Detection antibody 37 DEG C, 1h；Colour developing 10min；
This ELISA to the Salmonella paratyphi A in Salmonella, Salmonella typhimurium, that Salmonella of Argonne,
Moscow' paratyphi B, Thompson Salmonella, Bu Luokeli Salmonella, salmonella kentucky, Salmonella
Bacterium, salmonella dublin, salmonella typhi, Salmonella anatis, Arizona Salmonella all have cross reaction, correspondence
Detection limit P/N >=2.1, are respectively as follows: 1.5 × 106CFU/mL、1.5×106CFU/mL、6.3×106CFU/mL、1.3×
107CFU/mL、3.1×106CFU/mL、3.1×106CFU/mL、6.3×106CFU/mL、6.3×106CFU/mL、2.5×
107CFU/mL、2.5×107CFU/mL、6.3×106CFU/mL、2.5×107CFU/mL；With E.coli, E.coli O157:
H7, Enterobacter sakazakii, staphylococcus aureus, Listeria monocytogenes no cross reaction.
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