CN105777900B - Acidovorax avenae subsp monoclonal antibody and its hybridoma cell strain - Google Patents
Acidovorax avenae subsp monoclonal antibody and its hybridoma cell strain Download PDFInfo
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- CN105777900B CN105777900B CN201610270079.9A CN201610270079A CN105777900B CN 105777900 B CN105777900 B CN 105777900B CN 201610270079 A CN201610270079 A CN 201610270079A CN 105777900 B CN105777900 B CN 105777900B
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the monoclonal antibodies and its hybridoma cell strain of a kind of anti-melon bacterial Acidovorax avenae subsp.The monoclonal antibody is secreted by the hybridoma cell strain that deposit number is CGMCC NO.10413 to be generated, and the deposit number of the hybridoma cell strain is CGMCC NO.10413.Acidovorax avenae subsp monoclonal antibody of the invention can be used for detecting Acidovorax avenae subsp.
Description
Technical field
The invention belongs to bioengineering field more particularly to a kind of melon bacterial Acidovorax avenae subsps, specifically a kind of
Melon bacterial Acidovorax avenae subsp monoclonal antibody and its hybridoma cell strain.
Background technique
Melon fruit blotch is a kind of serious bacterial disease, and the late 1980s, the cause of disease was in the several states in the U.S.
Occur destructive outburst on watermelon and receive significant attention, since then, melon bacterial fruit blotch is worldwide spread.
Its cause of disease is acidophilus Pseudomonas oat kind watermelon subspecies (the Acidovorax avenae of Gram-negative bacteria
Subsp.citrulli, Aac), it is a kind of kind biography germ with high-destruction.Up to the present, have no to support completely
Resist the commercialized cultivation kind of the germ.
Acidovorax avenae subsp. citrulli is also known as Acidovorax avenae subsp, it can cause the cucurbitaceous plants illness such as watermelon, muskmelon, pumpkin, according to
Report, has also detected that the pathogen in the seed of the plants of Solanaceae such as tomato, eggplant, it can infect fruit, plant and seed,
Rely primarily on infected seed propagation.Infected seed can after planting make seedling illness, brown necrotic plaque occur.Fruit carry disease germs after adopting
Healthy fruit can be infected in storage transportational process, surface will appear water soaked spots, eventually lead to entire fruit rot, serious shadow
Ring melon yield.
The test paper for detecting Acidovorax avenae subsp is from U.S. Agdia company currently on the market, and country's report is less, and existing
There is antibody used in the immunity colloidal gold test paper strip in technology to be often made with monoclonal antibody as gold labeling antibody and Duo Ke
Grand antibody is used cooperatively as detection antibody, therefore low there is detection accuracy and cross reaction is high, test strips validity period is short etc.
Problem causes some influences to the precision of detection and accuracy.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of melon bacterial Acidovorax avenae subsps, and glue is immunized
Body gold Rapid detection test strip, this melon bacterial Acidovorax avenae subsp immune colloid gold Rapid detection test strip solve
The not high technical problem of test strips detection melon bacterial Acidovorax avenae subsp sensitivity in the prior art.
The present invention provides a kind of melon bacterial Acidovorax avenae subsp immune colloid gold Rapid detection test strips, including sample
Pad, in the gold-labelled pad that is closely coupled to the sample pad, the gold-labelled pad with the close-connected cellulose membrane of the gold-labelled pad
With the water absorption pad for being closely coupled to the cellulose membrane other end, matter is arranged in one end on the cellulose membrane far from gold-labelled pad
Control line, detection line be set on the cellulose membrane between nature controlling line and gold-labelled pad, wherein be provided in the gold-labelled pad with
The monoclonal antibody that colloidal gold couples, the hybridoma cell strain that the monoclonal antibody is CGMCC NO.10413 by deposit number
Secretion generates, and the detection line is made of monoclonal antibody, and the monoclonal antibody is CGMCC by deposit number
The hybridoma cell strain of NO.10413, which is secreted, to be generated.
Further, the nature controlling line is made of sheep anti-mouse antibody.
Further, in the monoclonal antibody that the colloidal gold couples, monoclonal antibody and colloidal gold are in mass body
Product is than being 24 μ g:1mL.
Further, the settable backboard to play a supportive role in the test strips back side.
Further, the test strips are fitted into a box body, and the position that the box body corresponds to sample pad is equipped with
Detection hole is equipped with observation window corresponding to the position of detection line and nature controlling line.
The present invention also provides a kind of monoclonal antibodies, by the hybridoma cell strain point that deposit number is CGMCC NO.10413
Secrete generation.
The present invention also provides a kind of hybridoma cell strain, deposit number is CGMCC NO.10413.
A kind of hybridoma cell strain of the invention, classification naming: anti-melon bacterial Acidovorax avenae subsp (Acidovorax
Avenae subsp.citrullii) hybridoma cell strain, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
In Bio-Centers (CGMCC), the address of China Committee for Culture Collection of Microorganisms's common micro-organisms center are as follows: court of Beijing
Positive area's North Star West Road 1 institute (institute of microbiology of the Chinese Academy of Sciences), the deposit date is on March 19th, 2015, deposit number CGMCC
NO.10413。
Testing principle of the invention and result deterministic process are as follows: if there is tested bacteria in sample, sample treatment liquid when measurement
It is added in sample pad, sample treatment liquid is mobile by the direction of sample pad, gold-labelled pad, nitrocellulose filter, water absorption pad, flows through golden mark
When pad, the monoclonal antibody coupled with colloidal gold in gold-labelled pad is redissolved, and it is driven to move to nitrocellulose filter, water absorption pad
It is dynamic, with the monoclonal antibody that colloidal gold couples immune complex, this immune complex stream can be formed in conjunction with the bacterium in sample
When to detection line, i.e., the monoclonal antibody of tested survey line is captured, formation bacterium+gold labeling antibody+antibody complex, in nitric acid
Detection line position on cellulose membrane shows red detection lines；When this immune complex flows through nature controlling line, i.e., by nature controlling line
Solid phase antibody is captured, and the Quality Control line position on nitrocellulose filter shows red Quality Control lines.
Positive sample not only shows detection line, but also display nature controlling line；' negative ' specimens do not have detection line, only show nature controlling line.
The present inventor's using watermelon plaque bacterial strain as immunogene studies have shown that being immunized Balb/c mouse, being isolated and purified
The hybridoma cell strain for being CGMCC NO.10413 to deposit number, the monoclonal antibody of hybridoma cell strain secretion after purification
Potency can reach 1:12800, can specificity, efficiently with 8 kinds of watermelon plaque bacterial strains ining conjunction with, it is sick with 3 kinds of Aac kindred plants
The equal no cross reaction of opportunistic pathogen.
The present invention compares with prior art, and technological progress is significant.The immune colloid of Acidovorax avenae subsp of the invention
Golden test strip sprays gold-labelled pad and detection line, experimental result due to using the pairing monoclonal antibody newly screened respectively
Show to Acidovorax avenae subsp detection specificity and sensitivity it is higher.
Detailed description of the invention
Fig. 1 is the monoclonal antibody SDS-PAGE electrophoresis of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
Figure.
Fig. 2 is the ascites of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp and purified antibodies effect in the embodiment of the present invention
Valence, wherein 2A indicates the potency of ascites antibody, and 2B indicates the potency of purified antibodies.
Fig. 3 is each antibody coupling colloidal gold pH of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
As a result.
Fig. 4 is each antibody coupling colloidal gold knot of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
Resultant result.
Fig. 5 is the structural schematic diagram of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention.
Fig. 6 is each antibody combination pairing knot of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
Fruit.
Fig. 7 is the colloidal gold strip sensitivity of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
As a result.
Fig. 8 is the colloidal gold strip detection 8 of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
As a result, wherein 8a indicates that test strips detect the testing result of 8 kinds of Acidovorax avenae subsps, 8b indicates test paper for kind Acidovorax avenae subsp and cross reaction
Item detects the testing result of 3 kinds of Aac kindred plant pathogens.
Specific embodiment
A specific embodiment of the invention is introduced below in conjunction with attached drawing:
Reagent used in the embodiment of the present invention and instrument are introduced first:
Main agents:
PEG, TWEEN-20 come from Sigma；BSA comes from Shanghai Shi Ze Biotechnology Co., Ltd；Colloidal gold solution comes from
Shanghai Jinbiao Bio-Tech Co., Ltd.；Nitrocellulose filter (NC film) 135S comes from Millipore；Sheep anti mouse, HRP- goat-anti
Mouse, goat-anti rabbit come spontaneous work bioengineering limited liability company.
Key instrument:
Microplate reader SpectraMax M2 is purchased from Molecular Devices；Nanodrop 2000C is purchased from Thermo
Scientific；Point sample instrument AD6010 is purchased from BIO-DOT；Scanner Phantom V9 is purchased from MICROTEK；Constant-temperature shaking incubator
SPH-100B is flat purchased from Shanghai generation；Protein purification instrument BioLogicTMLP 358BR5057 is purchased from BIO-RAD；Single purification work
Platform SW-SJ-2D type is purified purchased from Suzhou.
The preparation of 1 Acidovorax avenae subsp monoclonal antibody of embodiment
1. immunogene prepares
8 kinds of watermelon plaque bacterial strains first carry out Zengjing Granule in brain heart infusion fluid nutrient medium, after drawn in its solid medium
Line chooses single bacterium and falls within progress PCR identification after culture in brain heart infusion fluid nutrient medium, and measures OD value at 600nm, chooses growth
Very fast one plant as immune bacterial strain.Sterile saline centrifugation is resuspended 3 times, and adjusting bacteria concentration is 108CFU/mL。
2. the preparation process of monoclonal antibody
1) experimental animal: selecting 38 week old, and weight 20g or so, female Balb/c mouse are experimental animal.
2) immunization method: every mouse peritoneal injects 0.4mL immunogene, at interval of 2 weeks with same dosage booster shots one
It is secondary.
3) it takes a blood sample: taking a blood sample after 3 booster immunizations from tail vein, antiserum is measured using non-competing enzyme-linked immunization indirectly
Potency.No longer rise to potency, same amount immunogene is injected intraperitoneally, cell fusion is conventionally carried out after 3 days.
4) cell fusion: mouse to be fused plucks eyeball and takes blood, separates serum as positive control.The dislocation of mouse vertebra is put to death
Spleen is taken, is ground with syringe and obtains splenocyte suspension, is resuspended 3 times with serum free medium centrifugation respectively with SP2/0 cell, and
It is counted through blood counting chamber, supernatant is removed in centrifugation after mixing by 5:1, and 50%PEG is merged, by 105A/hole, which is laid on, has spread raising
In 96 orifice plates of confluent monolayer cells, hybridoma uses HAT Screening of Media, is placed in 37 DEG C, 5%CO2Incubator culture.
5) filtering hybridoma: when cell is paved with bottom hole 70%~80%, supernatant is taken to survey using indirect elisa method
It is fixed, strong positive hole is cloned again, 3~4 times repeatedly, until positive rate reaches 100%.Subcloning procedures use HT culture medium instead
Culture, indirect elisa method screening, each colony screening to positive hole expand culture after freeze in liquid nitrogen.
6) Antibody preparation: hybridoma being expanded and is cultivated, is injected in through the pretreated Balb/c mouse peritoneal of paraffin oil,
Every 2 × 106A hybridoma, 7~10 days mouse web portion protuberances, collects ascites, using saturated ammonium sulfate and Protein-G
Column affinitive layer purification ascites.
The hybridoma cell strain for filtering out the anti-Acidovorax avenae subsp monoclonal antibody of 4 plants of stably excretings, be respectively designated as 6F, 6D,
7E, 4F carry out subclass and subtype identification, measurement concentration, potency, purity respectively, and carry out to the monoclonal antibody of preparation special
Property verifying.
3. the subtype identification of monoclonal antibody
It, can by indirect ELISA measurement result by each Subclass of antibody of monoclonal antibody subtype identification kit measurement, hypotype
, subclass is IgG2a, and light chain subtype is κ.
4. the purifying of monoclonal antibody, purity and bioactivity
After ascites is first slightly mentioned with saturated ammonium sulfate, then purified with Protein G Sepharose affinity chromatography, will be prepared
Purifying gained antibody 6F, 6D, 7E, 4F measures concentration such as table 1 by Nanodrop 2000C analyzer, and 6F concentration is 12mg/mL,
6D concentration is 4mg/mL, and 7E concentration is 2mg/mL, and 4F concentration is 10mg/mL.
1 MAb concentration of table
| Antibody number | 6F | 6D | 7E | 4F |
| Concentration (mg/mL) | 12 | 4 | 2 | 10 |
With SDS-PAGE purity assay, 5% spacer gel, 10% separation gel, 120V voltage, electrophoresis to glue bottom, gel use
Coomassie Brilliant Blue dyeing, destainer decoloration.Fig. 1 is the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention
Monoclonal antibody SDS-PAGE electrophoresis.As shown, M is Marker, 1,2,3,4 be respectively 6F, 6D, 7E, 4F electrophoresis result, can
See, 4 plants of monoclonal antibodies have respectively one about 50KDa heavy chain, one about 25KDa light chain, without extra miscellaneous item
Band shows that foreign protein has been removed in ascites antibody, and purified antibodies purity is higher.
The step of ascites and after purification antibody titer measure is as follows:
1) by 108100 μ L, 37 DEG C of 2h are added in CFU/mL bacterial antigens in corresponding hole.
2) it empties liquid and pats dry residual liquid, cleaned 3 times with 230 μ L PBST washing lotions.
3) each Kong Zhongjia 220 μ L 3%BSA, 37 DEG C of closing 1h.
4) it empties liquid and pats dry residual liquid, cleaned 3 times with 230 μ L washing lotions.
5) each 100 μ L antibody of Kong Zhongjia (2mg/mL dilutes 1000 times), 37 DEG C of incubation 1h.
6) it empties liquid and pats dry residual liquid, each 230 μ L washing lotion of Kong Zhongjia is washed 3 times.
7) secondary antibody (sigma) that the HRP of 100 μ L is marked in each hole, is incubated at room temperature 1h.
8) it empties liquid and pats dry residual liquid, each 230 μ L washing lotion of Kong Zhongjia is washed 3 times.
9) each 100 μ L substrate of Kong Zhongjia, develop the color 15min, adds 100 μ L of terminate liquid and immediately in OD450nmReading.
As shown in Figure 2, ascites antibody potency is respectively 1:102400,1:102400,1:25600,1:51200 (Fig. 2A),
Monoclonal antibody (2mg/mL) potency is respectively 1:12800,1:6400,1:3200,1:6400 Fig. 2 B after purification).
5. monoclonal antibody specificity measures
To 8 kinds of Acidovorax avenae subsps and the nearly source phytopathogen combination situation of 3 kinds of Aac, as a result indirect ELISA measures 4 strain antibodies
It is shown in Table 2.
The specificity of 2 monoclonal antibody of table
Note: "+" is that can combine, and "-" is not combine
The hybridoma cell strain 6D4C5E9B9 of the Acidovorax avenae subsp monoclonal antibody Jing Guo above-mentioned identification is generated in 2015 3
It is preserved within 19th China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) (Chaoyang District, Beijing City north the moon
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101), classification naming is " to resist melon bacterium
Property Acidovorax avenae subsp (Acidovorax avenae subsp.citrullii) hybridoma cell strain ", deposit number CGMCC
No.10413。
Two, the determination of colloidal gold strip antibody optimum combination
Since different antibody combinations is affected to colloidal gold strip detection sensitivity and quality, it needs
Pairing optimum selecting is carried out to antibody used in the process of preparing colloidal gold strip.
1. the determination of the Optimal pH of each antibody coupling colloidal gold
Take 100 μ L colloidal gold solutions in 96 orifice plate, 8 holes respectively, and adjust pH to 5.5,6.0,6.5,7.0,7.5,
8.0,8.5,9.0, the monoclonal antibody that 5 μ L concentration are 2mg/mL is added in every hole.5min is reacted, 10 μ L 10% are added in each hole
NaCl solution reacts 5min, and observation solution colour changes, in triplicate.
PH value is followed successively by 5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 in the hole of each group from left to right in Fig. 3.By scheming
3 learn, for a group when pH is 7.5-8.0, colloidal gold color is most red in hole, and without coagulation, without fading or metachromatism, i.e. 6F are mono-
Anti- coupling colloidal gold optimum PH range is 7.5-8.0；B group when pH be 7.5 when, colloidal gold color is most red, and without coagulation, without colour fading
Or metachromatism, i.e. it is 7.5 that 6D, which is coupled colloidal gold Optimal pH,；C group when pH be 7.5 when, colloidal gold color is most red, and without coagulation,
Without metachromatism, i.e. it is 7.5 that 4F, which is coupled colloidal gold Optimal pH,；7E label failure.
2. the determination of the best combination amount of each antibody coupling colloidal gold
The optimal pH for adjusting each antibody coupling obtained in colloidal gold solution pH to step (1), respectively takes 100 μ L in 96 holes
In plate, each antibody is added by table 3, reacts 5min, 10 μ L10%NaCl solution are added in each hole, react 5min, observe solution colour
Variation takes that color is most red and antibody dosage is least for minimum binding capacity x μ g/mL, to guarantee that colloidal gold is complete in triplicate
In conjunction with antibody, then with x+x × 20% for best combination amount.
3 antibody of table and colloidal gold conjugate best combination amount
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Colloidal gold solution (μ L) | 100 | 50 | 100 | 100 | 100 | 100 | 100 |
| Antibody mass/gold solution volume (μ g/mL) | 0 | 0 | 5 | 10 | 15 | 20 | 25 |
| 10%NaCl (μ L) | 10 | 0 | 10 | 10 | 10 | 10 | 10 |
It is learnt by Fig. 4, when antibody mass/gold solution volume is 0 μ g/mL, colloidal gold in each group hole 1 after addition 10%NaCl
There is coagulation, color is graying by red, eventually becomes colourless；Each group hole 2 is that 50 μ L colloidal gold stostes compare.When antibody matter
Colloidal gold color, which successively reddens, when amount/gold solution volume increases to 25 μ g/mL by 5 μ g/mL, in each group hole deepens, wherein a group
When 6F antibody additive amount >=10 μ g/mL, color keep red is basically unchanged, and red in hole 3, i.e. 6F minimum binding capacity is 10 μ
G/mL, then its best combination amount is 12 μ g/mL；When b group 6D antibody additive amount >=20 μ g/mL, color change is smaller, and it is red in
Hole 3,4,5, i.e. 6D best combination amount are 24 μ g/mL；When c group 4F antibody additive amount >=15 μ g/mL, color keep red is substantially not
Become, and red in hole 3,4, i.e. 4F best combination amount is 18 μ g/mL.
3. the nano gold mark of antibody
Colloidal gold pH is adjusted to optimal pH, draws antibody by the best combination amount determined in step (2) with 40 μ L/min
Speed be added drop-wise in colloidal gold solution, while being stirred on magnetic stirring apparatus, continue to stir 30min after antibody adds, add
Enter the PBS solution containing 10%BSA of 1/10 volume of gold solution, stir 1h, moves into centrifuge tube 4000rpm and be centrifuged 10min, it is careful to inhale
It takes supernatant to new centrifuge tube, supernatant 13000rpm is centrifuged 25min, abandons supernatant, precipitating re-suspension liquid is with former colloidal gold solution 1/
The amount of 10 volumes is resuspended, and saves backup for 4 DEG C after mixing.It is resuspended, is uniformly mixed with the amount of former 1/10 volume of colloidal gold solution
It saves backup for 4 DEG C afterwards, if you need to save the long period, 0.3% sodium azide can be added.
4. the assembling and measurement of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp
Fig. 5 is the structural schematic diagram of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp of the present invention.
As shown in figure 5, the immune colloidal gold detection test paper strip 10 of Acidovorax avenae subsp includes: sample pad 14, gold-labelled pad 13, detection
Line 16, nature controlling line 15 and water absorption pad 11 use nitrocellulose filter 12 in the centre of water absorption pad 11 and sample pad 14, and nitric acid is fine
It ties up plain film and is also referred to as NC film.There is nature controlling line 15 and detection line 16, detection line 16 is close to sample pad side on nitrocellulose filter 12.
In addition, the immune colloidal gold detection test paper strip 10 of Acidovorax avenae subsp also has backing (not shown), for carrying above-mentioned sample
The structures such as product pad.
The gold labeling antibody prepared in step 3 is sprayed in the gold-labelled pad of multiple test strips respectively, is denoted as 6F-
Each antibody is diluted to 2mg/mL by Au-pad, 6D-Au-pad, 4F-Au-pad, is coated on nitrocellulose filter 12 and is made respectively
For Test line, i.e. detection line, also referred to as T line.Method for coating uses routine experiment method.It is tried using sheep anti mouse as gold-labelled monoclonal antibody
The Control line of paper slip, i.e. control line, also referred to as C line.It is anti-to gold-labelled pad and detection wire spraying by the combination in table 4
Body assembles test strips, by the Acidovorax avenae subsp SD01 normal saline dilution of culture to 108、107、106CFU/mL is measured, nothing
Bacterium physiological saline makees negative control, selects optimal antibody combination.
In table 4 in the column of gold-labelled pad one:
6F-Au-pad is represented using monoclonal antibody 6F as gold labeling antibody and in conjunction with colloidal gold and is sprayed at colloid gold test paper
In the gold-labelled pad of item.
6D-Au-pad is represented using monoclonal antibody 6D as gold labeling antibody and in conjunction with colloidal gold and is sprayed at colloid gold test paper
In the gold-labelled pad of item.
4F-Au-pad is represented using monoclonal antibody 4F as gold labeling antibody and in conjunction with colloidal gold and is sprayed at colloid gold test paper
In the gold-labelled pad of item.
Detection line file expression in table 4 does detection line using different antibody respectively.
4 antibody difference of table pairing assembling test strips
Fig. 6 is each antibody combination pairing result of the immune colloidal gold detection test paper strip of Acidovorax avenae subsp of the present invention.Such as Fig. 5 institute
Show, wherein a is 6F-6F combined result；B is 6F-6D combined result；C is 6F-7E combined result；D is 6F-4F combined result；e
For 6D-6F combined result；F is 6D-6D combined result；G is 6D-7E combined result；H is 6D-4F combined result；I is 4F-6F group
Close result；J is 4F-6D combined result；K is 4F-7E combined result；L is 4F-4F combined result.
It is from left to right respectively to use 10 in each group test strips in Fig. 68、107、106CFU/mL and sterile saline into
Row test as a result, as shown in Figure 5, f group sensitivity is up to 106CFU/mL, and non-false positive, effect are preferable；A, b, c, e, l group
Sensitivity is also up to 106CFU/mL, and non-false positive, but the colour developing of its T, C line is shallow compared with f group；D, g, j, k group T line is without colour developing,
For false negative；H, i group has false positive appearance, and the lower or possible colloidal gold of j, k, l group C line coating goat-anti rabbit concentration fails on label
It is mostly anti-that C line is caused not develop the color, to sum up, the preferable f group of antibody combination effect is selected, i.e. 6D is coupled colloidal gold and sprays as gold labeling antibody
It is applied to gold-labelled pad, 6D is sprayed at NC film and makees T line to make the immune colloidal gold detection test paper strip of Acidovorax avenae subsp of the invention.
5. pair f group, i.e. 6D is coupled colloidal gold as gold labeling antibody and is sprayed at gold-labelled pad, and 6D is sprayed at the group that NC film makees T line
It closes, carries out sensitivity determination experiment.
Cultured bacterium solution is successively diluted to 10 with physiological saline8、107、106、105CFU/mL, various concentration bacterium solution are equal
It is added dropwise in the sample pad using the test strips of f group monoclonal antibody combination production respectively with 50 μ L, is detected, use sterile physiological salt
Water is negative control, and in triplicate.
It chooses Acidovorax avenae subsp single bacterium to fall in culture medium, is placed in 36 DEG C of shaking table culture 12h, plate count calculates to obtain pure bacterium solution
Concentration is 4.1 × 108CFU/mL, as shown in Figure 7, it is 10 that concentration, which is added dropwise,8、107、106After CFU/mL bacterium solution, test strips T line is aobvious
Color is added dropwise 105CFU/mL bacterium solution T line illustrates test strips detection sensitivity up to 10 without colour developing6CFU/mL, reproducible results is equal three times
It is 106CFU/mL, Sensitivity Stability are preferable.
6. the immune colloidal gold detection test paper strip specificity experiments of pair Acidovorax avenae subsp
The immune colloidal gold detection test paper strip of Acidovorax avenae subsp is coupled colloidal gold as gold mark using monoclonal antibody 6D and resists at this time
Body is sprayed at gold-labelled pad, and monoclonal antibody 6D is sprayed at NC film and makees T line.3 plants of cultures are detected extremely with the test strips prepared
108The bacterium of CFU/mL simultaneously observes result.
A is that the immune colloidal gold detection test paper strip of Acidovorax avenae subsp in the embodiment of the present invention detects 8 kinds of Acidovorax avenae subsp knots in Fig. 8
Fruit；B is the immune colloidal gold detection test paper strip cross reaction result of Acidovorax avenae subsp in the embodiment of the present invention.
By Fig. 8 a it is found that 50 μ L concentration are 108CFU/mL 8 kinds of Acidovorax avenae subsps (99-5, xj112, PSLB1,00-1,
Plsb91, TW31, ATCC29625, SD01) after bacterium solution is added drop-wise to sample pad, T line develops the color；By Fig. 8 b it is found that other 3 plants close
After phytopathogen (NCPPB961, ATCC33996, ATCC19307) the same concentrations same volume sample-adding in source, T line is not shown
Color.The result shows that the test strips can 8 kinds of Acidovorax avenae subsps of single-minded detection (99-5, xj112, PSLB1,00-1, plsb91,
TW31, ATCC29625, SD01), specificity is preferably.
The present invention has filtered out four plants of monoclonal antibodies, and by experimental verification, it is best therefrom to have chosen resultant effect
Immunity colloidal gold test paper strip is made as gold labeling antibody and detection antibody in one plant of monoclonal antibody, i.e. 6D.Experiment shows such examination
Paper slip no cross reaction, has the characteristics that high sensitivity.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (2)
1. a kind of monoclonal antibody is secreted by the hybridoma cell strain that deposit number is CGMCC NO.10413 and is generated.
2. a kind of hybridoma cell strain, deposit number is CGMCC NO.10413.
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| CN111735953A (en) * | 2020-06-23 | 2020-10-02 | 南京农业大学 | Rapid immunodetection test strip for type II bacterial strains of melon bacterial fruit blotch and application of rapid immunodetection test strip |
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