CN102766209B - Monoclonal antibody of anti-perkinsus membrane protein, preparation method and application thereof - Google Patents

Abstract

The invention provides a monoclonal antibody of anti-perkinsus membrane protein (MOE). The monoclonal antibody of the anti-perkinsus membrane protein (MOE) is secreted by a hybridoma cell strain IAQ1101 of the anti-perkinsus membrane protein (MOE) with an accession number of CGMCC (China General Microbiological Culture Collection) No.6180. The invention also provides a preparation method and application of the monoclonal antibody. The monoclonal antibody of the anti-perkinsus membrane protein (MOE) is obtained by a genetic engineering technology and an immunological method; by labeling the monoclonal antibody with fluorescence, the immunofluorescence labeling technology is utilized to quickly and accurately detect infected perkinsus in shellfish, accordingly, a reliable immunological detection method is provided for the rapid identification of a port.

Description

Anti-monoclonal antibody, its preparation method and application of sending qin worm membranin

Technical field

The present invention relates to genetically engineered and field of immunology, specifically, relate to anti-monoclonal antibody, its preparation method and application of sending qin worm membranin (MOE).

Background technology

Sending qin worm (Perkinsus, or bat qin worm) is one of main pathogen affecting world's shellfishery development, and because it distributes extensively, harm seriously, is defined as and must reports hydrocoles disease by OIE (OIE).1997, China detected and sends qin worm in the young body of the clam from Philippines's import.Calendar year 2001 Liang Yubo etc., adopt mercaptoacetic acid salt culture medium (FTM) to cultivate and the method for tissue slice proves, clam son's the qin insect infection rate of sending is 20%-100%.Within 2008, the Ministry of Agriculture will send qin worm to list main infection clam son's parasite cause of disease in first.

In view of sending the severity of qin worm harm, carry out a large amount of research work both at home and abroad, at present, liquid mercaptoacetic acid salt culture medium (FTM) method is widely used in sending the infection qualification of qin worm, along with molecular biological development, there is the correlative study that utilizes regular-PCR, fluorescent PCR and LAMP to detect.But about sending the report of qin worm immunology detection aspect relatively less.

Summary of the invention

The object of this invention is to provide anti-monoclonal antibody, its preparation method and application of sending qin worm membranin (MOE).

In order to realize the object of the invention, the anti-monoclonal antibody of sending qin worm membranin (MOE) of one of the present invention, its anti-hybridoma cell strain IAQ1101(that sends qin worm membranin (MOE) that is CGMCC No.6180 by preserving number is S1005) secretion generation.

The present invention also provides the method for the above-mentioned anti-monoclonal antibody of sending qin worm membranin (MOE) of preparation: using the total DNA extracting from infect the shellfish tissue of sending qin worm as template, carry out pcr amplification taking 5 '-GGCTGATATCGGATCCTCCTCTTGTCCCACAGGGGATGC-3 ' and 5 '-CCGCAAGCTTGTCGACTTATGACGTAGGACATGTCGG-3 ' as primer, amplified production is inserted in expression vector, and transformed host cell, carry out protein expression with copying of host cell, then with the target protein immunity Balb/c mouse after purifying, get splenocyte and the murine myeloma cell fusion of immune mouse and prepare hybridoma, screening obtains the anti-hybridoma cell strain IAQ1101 that sends qin worm membranin (MOE), produce anti-monoclonal antibody of sending qin worm membranin (MOE) by its secretion.

In preceding method, pcr amplification system is counted with 25 μ L:

Pcr amplification condition is: 95 DEG C of 5min; 95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 40 circulations; 72 DEG C of 10min.

In preceding method, the expression vector preferably using is pET32a, and host cell is preferably e. coli bl21.

In preceding method, the murine myeloma cell of use is NS1, SP2/0-Ag14(abbreviation SP2/0) or X63Ag8.653.Preferably use SP2/0.

The present invention also provides the detection kit that contains described anti-monoclonal antibody of sending qin worm membranin (MOE).

The present invention also provides the described anti-monoclonal antibody of qin worm membranin (MOE) or the described detection kit of sending to send the application in the detection of qin worm shellfish: it adopts the method for immunofluorescence label, first antagonism sends the monoclonal antibody of qin worm membranin (MOE) to carry out fluorescent mark, then utilize in this fluorescently-labeled antibody test shellfish tissue slice or shellfish tissue homogenate thing, infect send qin worm.

The present invention further provides the anti-hybridoma cell strain IAQ1101(that sends qin worm membranin (MOE) is S1005), now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101, preservation date on June 5th, 2012, preserving number CGMCCNo.6180.

In order to obtain a large amount of anti-monoclonal antibodies of sending qin worm membranin (MOE), hybridoma is colonizated in mouse peritoneal, a large amount of ascites that results produce, external except containing required monoclonal anti in ascites, also contain more foreign protein, non-specific IgG and many proteolytic enzyme, easily make antibody inactivation, therefore ascites collect after purifying as early as possible, prevent degraded.

Adopt the anti-monoclonal antibody of sending qin worm membranin (MOE) after Western Blotting method purification Identification.The monoclonal antibody after purifying is carried out to SDS-PAGE, then according to ordinary method electrotransfer to nitrocellulose (NC) film, then seal-Jia Paiqin worm monoclonal antibody-sequence of operations such as colour developing.

Adopt Dot-ELISA (ELISA) to screen anti-monoclonal antibody of sending qin worm membranin (MOE).Be coated with elisa plate with the qin worm membranin (MOE) (being above-mentioned purpose albumen) of sending after purifying, add 1%BSA fluid-tight to close rear with PBS washing, then add the supernatant liquor (being the supernatant of the centrifugal rear collection of ascites) that contains anti-monoclonal antibody of sending qin worm membranin (MOE); After certain hour reaction, wash away unconjugated antibody; Add again the sheep anti-mouse igg of horseradish peroxidase-labeled; Taking horseradish peroxidase substrate (TMB) as chromogenic enzyme substrate.In supernatant liquor hybridoma oozy anti-send qin worm membranin (MOE) monoclonal antibody can with send qin worm membranin (MOE) combination, be then combined with ELIAS secondary antibody again, make substrate for enzymatic activity and aobvious blue, carry out detected result by microplate reader.

In addition, can be aided with microscopic examination, contribute to the infection to sending qin worm to position analysis.

The invention has the advantages that: the present invention utilizes genetic engineering technique and immunological method to obtain anti-monoclonal antibody of sending qin worm membranin (MOE), by monoclonal antibody is carried out to fluorescent mark, that utilizes immunofluorescence label technology fast, accurately to detect to infect in shellfish sends qin worm, for port Rapid identification provides reliable immunological detection method.

Brief description of the drawings

Fig. 1 is the pcr amplification result of target DNA fragment in the embodiment of the present invention 1; Wherein M is DNAMarker, and 1 is object fragment.

Fig. 2 is the SDS-PAGE electrophoresis result of target protein in the embodiment of the present invention 1; Wherein M is albumen Marker, and 1 is target protein.

Fig. 3 is the SDS-PAGE electrophoresis result of the anti-monoclonal antibody of sending qin worm membranin (MOE) of the purifying of the embodiment of the present invention 2; Wherein 1 is albumen Marker, 2 monoclonal antibodies that are purifying, and heavy chain is 58KD, light chain is 27KD.

Fig. 4 is the Western blotting analytical results of the anti-monoclonal antibody of sending qin worm membranin (MOE) of the present invention; Wherein M is albumen Marker, and 1 is monoclonal antibody of the present invention, and 2 is pET32a empty plasmid.

Fig. 5 is the qualification result of sending qin worm positive cell that utilizes immunofluorescence label technology for detection to cultivate; Wherein 1 positive cell brightness detecting result, 2 positive cell FITC fluoroscopic examination results, 3 negative contrast brightness detecting results, 4 negative contrast FITC fluoroscopic examination results.

Fig. 6 is that the present invention utilizes the infection of immunofluorescence label technology for detection to send the qualification result of the young paraffin section of clam of qin worm; Wherein 1 is Ha Zai gill tissue paraffin section brightness detecting result, 2 is the paraffin section FITC of Ha Zai gill tissue fluoroscopic examination result, 3 is the young mantle tissue paraffin section of clam brightness detecting result, 4 is the young mantle tissue paraffin section of clam FITC fluoroscopic examination result, 5 is the young viscera tissue paraffin section of clam brightness detecting result, and 6 is the young viscera tissue paraffin section of clam FITC fluoroscopic examination result.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.

Embodiment 1 sends the acquisition of qin worm membranin (MOE)

1.1 design of primers

According to the disclosed sequence of ACCESSION No.EF632302 in the state-run bioinformation of U.S. center NCBI gene pool (GenBank), at open reading frame two ends design primer, primer sequence is:

CB5D4UP:5 '-GGCTGATATC gGATCCtCCTCTTGTCCCACAGGGGATGC-3 ' (underscore place is BamHI restriction enzyme site)

CB5D4LP:5 '-CCGCAAGCTT gTCGACtTATGACGTAGGACATGTCGG-3 ' (underscore place is SalI restriction enzyme site)

The preparation of 1.2 primers

After synthetic primer, be 10 μ mol/l by primer dilution, upstream and downstream primer mixes according to the ratio equal-volume of 1:1, and after packing ,-20 DEG C save backup.

1.3 send the extraction of qin worm DNA

Choose to infect and send the unsound of qin worm or dead philippine clam whelp just, separate gill tissue, with commercial kit (Dneasy Blood and Tissue kit) the extraction DNA of QIAGEN company.

The pcr amplification of 1.4 goal gene

PCR reaction system is as follows:

Pcr amplification condition is: 95 DEG C of 5min; 95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 40 circulations; 72 DEG C of 10min.

After reaction finishes, detect with 1% agarose gel electrophoresis, visible object band 357bp, size conforms to expection, and electrophoresis result is shown in Fig. 1.

The structure of 1.5 recombinant expression plasmids

PCR product after reclaiming is inserted in PMD19-T simple carrier and is built into recombinant clone plasmid, be connected with the same pET32a expression vector of cutting through these two enzyme enzymes after SalI double digestion through BamHI, be built into recombinant expression plasmid, transform BL21, the single bacterium colony of picking white shakes bacterium, and bacterium liquid is carried out to PCR and order-checking qualification.Order-checking qualification result show, the nucleotide sequence of object fragment is as shown in Seq ID No.1.

The abduction delivering of 1.6 target proteins

Shake in a large number bacterium to being accredited as positive bacterium liquid through PCR and order-checking, until bacterium liquid, OD600 is about at 0.6 o'clock, adds the IPTG of 10 μ mol to continue induction 4h.Collect the bacterium liquid after induction, ultrasonication, collects respectively supernatant and precipitation and carries out SDS-PAGE electrophoretic analysis, and electrophoresis result shows that the target protein of expressing is present in supernatant, according to Ni-Agarose His label protein purification kit operation instruction operation, get supernatant and cross Ni post and carry out protein purification.Purifying protein carries out SDS-PAGE electrophoresis, and electrophoresis result is shown in Fig. 2.

The acquisition of the anti-monoclonal antibody of sending qin worm membranin (MOE) of embodiment 2

1.1 purifying send qin worm membranin (MOE) immune mouse

Send qin worm membranin (MOE) and the Freund's complete adjuvant equal-volume of purifying are mixed, and fully concussion mixes emulsification, grinds to form water in oil chyle shape (add dropping in to be difficult for spreading on the water surface and be droplet-shaped and show to reach water in oil state).

1) initial immunity: specificity sends qin worm membranin (MOE) 1 ~ 80 μ g to add the subcutaneous multi-point injection mouse of Freund's complete adjuvant (general 0.25 ~ 1mL, 0.2mL/ point);

2) immunity for the second time: post dose reduced by half in 1 week, added the subcutaneous multi-point injection mouse of Freund's incomplete adjuvant;

3) for the third time immunity: within 3 days, post dose is the same, add the subcutaneous multi-point injection mouse of Freund's incomplete adjuvant (5 ~ 7 days afterwards blood sampling survey its tire, detect immune result);

4) booster immunization: after 1 ~ 2 week, dosage 80 ~ 500 μ g are advisable, subcutaneous multi-point injection mouse;

5) booster immunization is after 3 days, and aseptic extracting spleen cell, prepares splenocyte suspension.

The preparation of 1.2 immune spleen cells

Last booster immunization is after 3 days, and aseptic extracting spleen cell, prepares splenocyte suspension, and concrete operations are as follows:

1) dislocation method is put to death mouse;

2) mouse is placed in after 75% alcohol soaking disinfection, cuts off its belly with sterile scissors, take out spleen, be placed in the aseptic stainless steel of placing containing the plate of a small amount of RPMI-1640 nutrient solution and shine on the net, count after grinding to form cell suspension with syringe nook closing member;

3) collect splenocyte suspension on ice, the centrifugal supernatant that goes;

4) will precipitate with fresh RPMI-1640 nutrient solution recentrifuge washing;

5) add fresh RPMI-1640 nutrient solution;

6) finally count splenocyte.

1.3 myeloma cells' preparation

The myeloma cell strain that at present conventional Balb/c mouse produces has: NS1, SP2/0-Ag14(are called for short SP2/0), X63Ag8.653 etc.In the present embodiment, use SP2/0.

Myeloma cell is stored in-196 DEG C of liquid nitrogen containers, when test, is recovered, breed, go down to posterity.48-36h before fusion, Ying Xianyong spreads cultivation containing the substratum of 15 μ g/mL8-azaguanine, gets approximately 1 × 10 ⁷~ 5 × 10 ⁷the myeloma cell of/mL logarithmic phase, in room temperature centrifugal (400r/min) 10min, by the resuspended precipitation of 2.5% foetal calf serum (FCS)-RPMI-1640 centrifugal, resuspended mix stand-by, and last counting.

The preparation of 1.4 feeder cell

In the time preparing feeder cell, must guard against the digestion organs of punctures animal, otherwise the cell that obtains has severe contamination.Concrete operations are as follows:

1) mouse, 75% alcohol-pickled sterilization 10min are put to death in dislocation;

2) with operating scissors, mouse web portion is cut off to an osculum, strip off skin, exposes abdominal cavity;

3) with syringe, 4 ~ 5mL serum-free RPMI-1640 nutrient solution is injected to abdominal cavity, with the soft massage of finger belly, still use this syringe pumpback abdominal cavity liquid, move into centrifuge tube;

4) the centrifugal 10min of 1000/min, removes supernatant;

5) with the RPMI-1640 nutrient solution suspendible containing 20% calf serum (NCS), adjust cell count 4 × 10 ⁵/ mL;

6) with 1mL suction pipe, cell is added to 96 well culture plates, every hole 0.05mL(is containing 20,000 cells), put into 37 DEG C, 5%CO ₂incubator is cultivated, and gained feeder cell can be for cytogamy and cloning.

1.5 cytogamy

The basic step of cytogamy is after myeloma cell is mixed with immunocyte, makes two kinds of cells merge each other under the effect of PEG, and the cell after merging is suitably diluted, and is placed in respectively containing the culture plate of feeder cell and cultivates.Concrete operations are as follows:

1) myeloma cell through anticipating is mixed in the ratio of 1:5 with immune spleen cell, the full nutrient solution that toos many or too much for use in 50mL plastic centrifuge tube is washed 1 time, 1200rpm/min, 8min;

2) abandon supernatant, with the dropper residual liquid that exhausts, in order to avoid affect PEG concentration; At the bottom of attack centrifuge tube, make cell precipitation loosening slightly gently;

3) by the 50%PEG(molecular weight 4000 being incubated in 37 DEG C of water-baths) 0.5mL slowly splashes in centrifuge tube, in 1min, adds, shake while dripping;

4) add the incomplete nutrient solution of preheating, add respectively 1mL, 2mL, 3mL, 4mL, 5mL and 10mL every 2min, stop PEG effect;

5) centrifugal, 800rpm/min, 6min;

6) abandon supernatant, add the about 6mL of RPMI-1640 substratum (Gibco, the U.S.) containing 20% calf serum, suspendible gently, must guard against firmly piping and druming, in order to avoid the cell merging is scattered;

7) with aseptic straw, cell suspension after merging is added and contained in 96 orifice plates of having inoculated in advance feeder cell, 100 μ L/ holes, put into 37 DEG C, 5%CO ₂incubator is cultivated.

1.6HAT screens hybridoma

Generally merging after 24h, adding xanthoglobulin-methotrexate-thymidine (HAT, Sigma, the U.S.) to select nutrient solution.When use, in 1mL fused cell suspension, add 50mL to contain the complete culture solution of 20% calf serum.Splenocyte can only be survived several days under culture condition in vitro, does not affect Growth of Hybridoma Cell; And myeloma cell is due to cannot purine biosynthesis cyclization thymus pyrimidine methyl in the time that folic acid inhibitor aminopterin exists and can not synthetic DNA, simultaneously again because it is HGPRT-and TK-defective type, cannot utilize HGPRT and TK in nutrient solution to carry out synthetic DNA, therefore myeloma cell cannot be survived in this nutrient solution.And finally can in HAT nutrient solution, survive only for hybridoma, because it has the karyomit(e) of two kinds of parental cells, can produce the HGPRT that original splenocyte has, from myeloma cell, obtain again the ability of long-term survival and reproduction in cell culture medium.

Use HAT to select nutrient solution maintain after two weeks, use HT nutrient solution instead, then maintain one week, general nutrient solution used instead.

Cultivate by selectivity in the hybridoma cell line obtaining, only minority can be secreted for immunogenic specific antibody.Generally at the bottom of hybridoma is covered with hole when 1/10 area, can start the anti-antibody of sending qin worm membranin (MOE) of detection specificity, filter out the hybridoma cell line of required hypersecretion, and finally to obtain the positive anti-hybridoma cell strain IAQ1101(that sends qin worm membranin (MOE) be S1005), preserving number CGMCC No.6180.

The cloning of 1.7 hybridomas

Fused cell is clonal growth, and after limiting dilution, should have 36% hole is 1 cells/well.Detect and filter out high antibody secretory pit by ELISA, cell in hole is carried out to cloning again.The principle of cloning is should carry out as early as possible cloning for detecting the anti-hybridization clone who sends qin worm membranin (MOE) antibody positive, even if the hybridoma of cloning also needs regular cloning again.Concrete operations are as follows:

1) prepare feeder cell suspension (preparing with the feeder cell before merging);

2) counting of positive porocyte, and regulate cell count 1 ~ 5 × 10 ³/ mL;

3) get 130 cells and add 6.5mL to contain feeder cell complete culture solution (i.e. 20 cell/mL), add A, B, C tri-rows with 100 μ L/ holes, i.e. 2, every hole cell; Remaining 2.9mL cell suspension is added the complete culture solution of 2.9mL containing feeder cell, and cell count is 10/mL, adds D, E, F tri-rows with 100 μ L/ holes, i.e. 1, every hole cell; Remaining 2.2mL cell suspension is added the complete culture solution of 2.2mL containing feeder cell, and 5/mL of cell count, with 100 μ L/ holes, adds G, H two rows, i.e. 0.5, every hole cell;

4) cultivate after 4 ~ 5 days, add complete culture solution 200 μ L/ holes;

5), 8th ~ 9 days time, naked eyes visible cell clone, carries out anti-detection of sending qin worm membranin (MOE) antibody in time.

1.8 utilize ascites to prepare anti-monoclonal antibody of sending qin worm membranin (MOE)

Adopt mice celiac inoculation, at the anti-hybridoma cell strain IAQ1101 that sends qin worm membranin (MOE) of mouse Inoculation, preparation ascites.Concrete operations are as follows:

1) Balb/c mouse peritoneal injecting fluid paraffin, 0.5mL/ is only;

2) after 1 ~ 2 week, to above-mentioned mouse peritoneal injection 1 × 10 ⁶individual hybridoma/only;

3) inoculating cell can produce ascites after 7 ~ 14 days, and now mouse web portion obviously swells, and hand touches fluctuation, by No. 16 syringe needles collection ascites;

4) by centrifugal the ascites 1500r/min collecting 30min, purifying in time.

1.9 anti-purifying of sending qin worm membranin (MOE) monoclonal antibody

Adopt Protein G-Sepharose affinity chromatography to carry out antibody purification, concrete operations are as follows:

1) balance: steady to baseline with binding buffer liquid (20mmol/L PBS, pH is 7.0) balance proteinG affinity column;

2) loading: by ascites sample upper prop, collect stream and wear liquid; Stream is worn to liquid upper prop again, continue balance steady to baseline;

3) wash-out: add elution buffer wash-out, collect elution peak, SDS-PAGE detects purity;

4), with 0.01M, the elution peak that the PBS liquid dialysis of pH7.2 is collected, makes the antibody after purifying be stored in 0.01M, in the PBS liquid of pH7.2;

5) concentration of monoclonal antibody after use protein quantification detector mensuration purifying;

6) antibody purity after SDS-PAGE detection purifying, antibody applied sample amount is 8 μ g, electrophoresis result is shown in Fig. 3.

Result shows, in ascites, the anti-concentration of qin worm membranin (MOE) antibody of sending of specificity is compared with the height in antiserum(antisera), and purification effect is good.

Embodiment 3 measures tire (the ELISA detection method) of anti-monoclonal antibody of sending qin worm membranin (MOE)

1) coated: add 2 μ g/ml antigens (specificity that is preparation in embodiment 1 is sent qin worm membranin (MOE)), 100 μ l/ holes, 4 DEG C are spent the night, washing lotion washing 3 times.

2) sealing: add 150 μ l/ hole confining liquids, 37 DEG C after 2 hours, wash 3 times, pat dry.Put 4 DEG C of Refrigerator stores for subsequent use.

3) add testing sample:

A. detect for serum/antibody/titer of ascites, first hole 1:1000 dilution, down, successively with the gradient doubling dilution of 1:3, hatches 30min for 37 DEG C, washes plate 4 times, pats dry.

B. detect for cell conditioned medium, get cell conditioned medium 100 μ l, add in corresponding enzyme plate, hatch 30min for 37 DEG C, wash plate 4 times, pat dry.

4) add two anti-: after the sheep anti-mouse igg (IgG special two is anti-) of getting horseradish enzyme labelling doubly dilutes by 1:5000,100 μ l/ holes, hatch 20 to 30min for 37 DEG C, wash 4 times, pat dry.

5) colour developing: dilution 20 × TMB to 1 × TMB, adds 37 DEG C of colour developing 15-30min by 100 μ l/ holes.

6) stop: add stop buffer (2M H ₂sO ₄) 50 μ l/ holes.

7) reading: measure each hole OD value with the mono-wavelength of 450nm, be limited to be greater than 2.1 with the ratio (P/N) of negative control hole OD value, as being judged as the positive or determining the stagnation point of tiring.

When note: a. screening positive cell, P/N>2.1 is judged to be positive cell; The cell hole of 1<P/N<2.1 detects after strengthening coated concentration again, and P/N>2.1 is still judged to be the positive; B. tire and represent taking P/N as 2.1 serum maximum dilution multiple.

The anti-monoclonal antibody of sending qin worm membranin (MOE) of embodiment 4 immunoblotting assays

Adopt Western blotting method, send qin worm membranin (MOE) to carry out SDS-PAGE electrophoresis to the specificity of purifying, according to ordinary method by albumen electrotransfer to nitrocellulose (NC) film, 1%BSA seals; Repetitive scrubbing NC film 3 times, adds the anti-monoclonal antibody of sending qin worm membranin (MOE) of preparing in embodiment 2, hatches 2 hours for 37 DEG C; Repetitive scrubbing NC film 3 times, adds sheep anti mouse-HRP again, hatches 1 hour for 37 DEG C; Repetitive scrubbing NC film 3 times; Add the colour developing of DAB nitrite ion; Tap water adds stop buffer after rinsing.Result shows, in embodiment 2, the monoclonal antibody of preparation sends qin worm membranin (MOE) to present specific reaction zone to the specificity being transferred on NC film, the results are shown in Figure 4.

Embodiment 5 utilizes immunofluorescence analysis detection to infect the shellfish tissue slice of sending qin worm

1.1 send the mark of qin worm antibody FITC

1) by the monoclonal antibody hybrid reaction 2 hours of preparation in FITC and embodiment 2;

2) above-mentioned reacted mixture is crossed to Sephadex G-25 post, remove free composition;

3) utilize nucleic acid-protein analysis-e/or determining concentration and F/P value.The traget antibody concentration recording is 2.11mg/mL, and F/P value is 6.76.

1.2 positive cell immunofluorescences detect

1) that gets that 200 μ L cultivate sends qin worm positive cell, and the centrifugal 5min of 5000rpm adds the resuspended precipitation of PBS of 200 μ L, more centrifugal, so repeatedly washes after 3 times, adds 200 μ L PBS resuspended for subsequent use.

2) get 20 μ L cell suspensions and be added on poly-lysine slide glass, examine under a microscope cell density, if cell density is excessive, redilution cell, does not occur overlapping to observation of cell under microscope.

3) room temperature is dried slide.

4) add the fixing slide 10min of 4% paraformaldehyde 50 μ L.

5) PBS develops a film 3 times, and after each 15min, room temperature is dried.

6) dyeing: the fluorescence antibody that 1:100 ~ 1:25600 of dropping 50 μ L doubly dilutes, slide is put in wet box, prevent from being dried, lucifuge, hatches 30min for 37 DEG C.

7) PBS develops a film 3 times, and after each 15min, room temperature is dried.

8) mounting: drip 10 μ L mountant, build cover glass, prevent bubble, at fluorescence microscopy Microscopic observation, detected result is shown in Fig. 5.

The making of 1.3 shellfish tissue paraffin section des

1) fixing: to get fresh shellfish, extraction postgenome PCR detection is accredited as to positive clam son and carries out paraffin section making, all get the gill, mantle and internal organ related tissue for every, finishing tissue block, blot rear 10% formaldehyde solution of putting into rapidly with filter paper and fix 24h, the consumption of formaldehyde is 30 ~ 50 times of tissue block volume of being fixed.

2) washing: gauze on bottleneck cover, secure with rubber band, if tissue block can be wrapped tissue block to put into together wide-necked bottle with gauze more at most, tap water connects rubber tubing and inserts a bottle end, adjust flux slowly flows out water at the bottom of bottle.

3) dehydration: soak time, depending on tissue size, is generally 4 ~ 12h in alcohol, and soaking the ethanol concn gradient using is 30% ethanol → 50% ethanol → 70% ethanol → 80% ethanol → 90% ethanol → 95% ethanol → 100% ethanol; Shorter in raw spirit.

4) transparent: dimethylbenzene is transparent, object is that the raw spirit in tissue block is cemented out, for waxdip is prepared.Transparent front elder generation is with dimethylbenzene-raw spirit transition of 1:1, prevents that dimethylbenzene from causing contraction to tissue, then uses dimethylbenzene transparent, and the time is generally 2 ~ 3h.

5) embedding: the first dimethylbenzene-paraffin transition through 1:1 of tissue block after transparent, then immerse wax cup, and in each wax cup, the residence time is no more than 30min, and above step is all carried out under paraffin melting state, strictly temperature is controlled at lower than 56 DEG C.After paraffin immerses tissue block completely, carry out the embedding-paraffin having dissolved is poured in small paper box, to put into cold water cooling in gripping rapidly, is frozen into piece.

6) section: embedded wax stone is fixed on slicing machine, thinly slices, be generally 5 μ m, be put in the water of heating and plate, make it to adhere on silication slide glass, put in 45 DEG C of thermostat containers and dry.

1.3 direct immunofluorescence antibody detection methods detect shellfish tissue slice

1) dewaxing of paraffin section routine, hydration process: use successively dimethylbenzene 10min, dehydrated alcohol 3min, 90% ethanol 3min, 70% ethanol 3min dewaxes and hydration process to paraffin section.

2) paraffin section collagenase treatment: tissue slice is put in 0.125mg/ml PRONASE A solution and digests (37 DEG C, half an hour), then clean section 5min with 0.2% glycine solution, termination reaction, wash 5min with distilled water, then in 4 DEG C, in 0.4% formaldehyde after fixing 5min, then wash 5min with PBS, last natural air drying.

3) dyeing: the section of handling well is dripped to the fluorescence antibody of 50 μ l dilutions, slide is placed in wet box, prevent from being dried, 37 DEG C of lucifuges are hatched 30min.

4) PBS develops a film 3 times, each 15min, room temperature natural air drying.

5) mounting: drip 10 μ l mountant, build cover glass, prevent bubble, at fluorescence microscopy Microscopic observation, take pictures, detected result is shown in Fig. 6.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (5)

1. anti-monoclonal antibody of sending qin worm membranin, is characterized in that, its anti-hybridoma cell strain IAQ1101 secretion of sending qin worm membranin that is CGMCC No.6180 by preserving number produces.

2. contain the detection kit of anti-monoclonal antibody of sending qin worm membranin described in claim 1.

3. described in claim 1, described in anti-monoclonal antibody of sending qin worm membranin or claim 2, detection kit is sent the application of qin worm in detecting shellfish.

4. application according to claim 3, it adopts the method for immunofluorescence label, first antagonism sends the monoclonal antibody of qin worm membranin to carry out fluorescent mark, then utilize in this fluorescently-labeled antibody test shellfish tissue slice or shellfish tissue homogenate thing, infect send qin worm.

5. the anti-hybridoma cell strain IAQ1101 that sends qin worm membranin, its preserving number is CGMCC No.6180.