CN106526179B - A kind of PVY substances virus colloidal gold Rapid detection test strip - Google Patents

Abstract

The invention discloses a kind of PVY substances virus colloidal gold Rapid detection test strips, are made of sample pad, colloidal gold pad, NC films, absorbent filter and backing；The PVY specific antibodies of colloid gold label are coated in the colloidal gold pad, which is secreted by hybridoma cell strain BALB/c 15 8 generates；It is provided with detection line and control line on the NC films, the specific antigen of PVY is coated in detection line；Secondary antibody is coated on control line.Detection of the test strips especially to the PVY of Tobacco-growing areas in Guangdong, it is quick, sensitive, accurate, at low cost, easy to operate, realize primary sample, multisample diagnoses simultaneously, it is very suitable for carrying out live primary dcreening operation to batch samples, has very much application value, popularizing application prospect good in actual production.

Description

A kind of PVY substances virus colloidal gold Rapid detection test strip

Technical field

The invention belongs to pathogen detection technique fields.It is quickly examined more particularly, to a kind of PVY substances virus colloidal gold Test paper slip.

Background technology

Marmor upsilon (Potato virus Y, PVY) is to infect one of main virus of tobacco.The virus that it causes Disease leads to the huge economic loss of tobacco industry every year.Quickly and accurately qualitative detection correlated virus be the upper prevention and control disease of production, Reduce one of the important means of loss.

In scientific research, the detection method of plant virus mainly has biology detection, and (symptom type distinguishes, differentiates and post It is main etc.), electron microscopy, serological detection method (Enzyme-linked Immunosorbent Assay react (Enzyme linked immunosorbent Assay, ELISA) etc.) and molecular biological assay (including round pcr, Nucleic Acid Probe Technique etc.) etc..Isolated viral is according to electricity Although mirror is to diagnose viral effective means, high specificity, but very time-consuming and laborious, needs technical professional, uncomfortable Conjunction is promoted the use of.Serology test is relatively time-consuming, laborious.Although the methods of immunofluorescence, ELLSA have it is micro, special, Fast and accurately advantage, but need more complete test apparatus and experienced technical staff to operate and judging result, The a collection of sample whole flow process of detection needs 4~8 hours, is difficult to accomplish for grass-roots work place.The diagnosis of PCR and nucleic acid probe With greater need for having special instrument and drug, technology content is very high, is generally adapted only to laboratory diagnosis or research application, it is difficult in base Layer is promoted.Therefore, there is an urgent need to establish a kind of viral diagnosis side that is simple, quick, sensitive, cheap and being suitable for base's application Method.

Colloidal gold in 1971 starts to be used for immunohistochemistry as marker.Faulk etc. applies Electronic Speculum immune colloid gold Decoration method observes salmonella.Many scholars further confirm that colloidal gold can stablize promptly adsorbed proteins, and protein Bioactivity is without substantially changeing.It can be used as probe to carry out cell surface and intracellular polysaccharide, protein, polypeptide, antigen, swash The large biological molecules such as element, nucleic acid are accurately positioned, and can be used for daily immunodiagnosis, carry out immunohistochemical localization. Colloidal gold solution refers to aurosol of the dispersed phase particles diameter between l~150nm, belongs to multiphase heterogeneous system, and color is in Salmon pink is to aubergine.Immune colloidal gold chromatography technology (Gold immunochromatography assay, GICA) is used as one Kind new immunological detection method is that one kind for being set up in immuno-gold labeling technical foundation the 1980s is new The unique diagnostic techniques of type.This technology is by colloidal gold-labeled method, immunoassay technology, Chromatographic techniques, list (more) gram A variety of methods such as grand antibody technique and new material technology organically combine, and have simple, quick, accurate, pollution-free, operation It the advantages that simply and without special installation, has been obtained in medicine, the animal and plant quarantine, each field of food safety supervision increasingly extensive Using.Early 1990s, Gold-immunochromatography assay diagnostic techniques initially entered commercial applications to mid-term.Utilize this The colloidal gold fast detecting test paper strip of kind of technological development have it is easy to operate, quick, can single part of detection, convenient for preserving, be not required to spy The advantages that different equipment, (clinic) can carry out primary dcreening operation to target cause of disease at the scene, save a large amount of human and material resources.In medical domain, This technology except for the antigen of hormone, infectious disease pathogens and antibody, bacterium, parasite detection in addition to, also developed to poison The detection of the small-molecule substances such as product.The specimen types of detection also contemplated serum, blood plasma, whole blood, urine, excrement and saliva etc., show Show wide foreground and huge application value.

Currently, colloidal gold strip is mainly used in medical domain and animal and veterinary field, plant virus aspect at home Seldom see.In agriculture and animal husbandry field, the product of rarer maturation more appears in relevant scientific research project.Sun Yan etc. (2011) cucumber bacterial angular leaf spot (Pseudomona.s.syringae has been carried out using colloidal gold technique Pv.Lachrymans quick detection research).The country is related to the commercial colloidal gold strip of tobacco virus detection very It is few.External commercialization test strips are very expensive, and price is unsuitable for the upper large quantities of tobacco seedlings of production at 50~60 yuans/ Detection uses.

Invention content

The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of Tobacco-growing areas in Guangdong PVY substance virus colloidal gold Rapid detection test strips.Core technology is that exploitation is directed to tobacco in seedling stage mainly viral marmor upsilon The substance colloidal gold colloidal gold detection test paper strip of (Potato virus Y, PVY), makes tobacco grower easy to operate, and diagnosis is promptly and accurately.

The object of the present invention is to provide the hybridoma cell strain BALB/c-15-8 that one plant can generate the special monoclonal antibodies of PVY.

Another object of the present invention is to provide a kind of PVY substances virus colloidal gold Rapid detection test strip.

Another object of the present invention is to provide the application of the PVY substances virus colloidal gold Rapid detection test strip.

Above-mentioned purpose of the present invention is achieved through the following technical solutions：

The hybridoma cell strain BALB/c-15-8 of the special monoclonal antibody of one plant of generation PVY, was preserved on June 30th, 2016 China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC No.12677, preservation address： Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

Meanwhile the special monoclonal antibodies of PVY generated by above-mentioned hybridoma cell strain BALB/c-15-8 secretions are also in the guarantor of the present invention Within the scope of shield.

In addition, application of the above-mentioned PVY specific antibodies in terms of preparing PVY viral diagnosis preparation or product is also the present invention's Within protection domain.Preferably, the PVY viruses refer to the PVY viruses of Tobacco-growing areas in Guangdong.

Specifically, the PVY substance virus colloidal gold Rapid detection test strips prepared by above-mentioned PVY specific antibodies, also at this Within the protection domain of invention.

Preferably, the PVY substances virus colloidal gold Rapid detection test strip is by sample pad, colloidal gold pad, NC films, water suction Filter paper and backing composition；The sample pad, colloidal gold pad and NC films are incorporated in successively according to sequence from left to right, from top to bottom On backing the same face, the end of colloidal gold pad and NC films is overlapped；The absorbent filter is incorporated in the other end of NC films；The colloid It is coated with the PVY specific antibodies of colloid gold label in gold pad, detection line (T lines) and control line (C lines) are provided on the NC films, And detection line (T lines) is located at the position between colloidal gold pad and control line (C lines)；PVY viruses are coated in detection line (T lines) Specific antigen；It is coated with secondary antibody on control line (C lines).

Above-mentioned PVY substances virus colloidal gold Rapid detection test strip has application well in the context of detection of PVY viruses Foreground, in particular for the PVY viruses of Tobacco-growing areas in Guangdong, detection sensitivity can reach 10^-2mg/mL。

The present invention utilizes A competitive inhibition method, successfully constructs PVY substance test strips.Its basic principle is as follows：

The PVY specific antibodies of colloid gold label are adsorbed on bonding pad (i.e. colloidal gold pad), the specific antigens of PVY viruses with On ribbon is fixed at the p-wire (T lines) of nitrocellulose membrane, secondary antibody is fixed at the control line (C lines) of nitrocellulose membrane. It after sample to be checked is added in the sample pad of test strips one end, moves forward through capillary action, dissolves the colloid on bonding pad When reacting to each other after gold label specific reagent, then being moved to fixed antigenic domains, the conjugate of object to be checked and golden labelled antibody It specifically binds therewith again.

When sample to be tested contains PVY viruses, it is mutual with the specific antibody for the colloid gold label being dissolved in sample pad Reaction；When being moved to fixed antigenic domains again, not enough gold labeling antibodies and fixed antigen-reactive do not have at T lines It is the positive to have the appearance of rufous lines, experimental result.When free gold labeling antibody or gold labeling antibody complex logistics is through at C, with There is rufous quality control band in two anti-bindings at this.

When sample to be tested does not have PVY viruses, it does not react with the colloidal gold labeled monoclonal antibody being dissolved on bonding pad； When being moved to fixed antigenic domains again, there are enough gold labeling antibodies and fixed antigen-reactive, and is trapped and is gathered in T lines On, rufous band can be observed by the naked eye, experimental result is feminine gender.

The invention has the advantages that：

The present invention is directed to the test strip that plant virus constructs PVY substance viruses for the first time, innovative very strong, exploitation PVY substance virus Rapid detection test strips utilize colloidal gold immunochromatographimethod technology, combine the original of chromatography and immune response Reason, realizes the purpose of quickly and accurately qualitative detection cause of disease, can field quickly, detect sample on a large scale, detection knot Fruit is accurate, reliable.Compared with ELISA method, test strips of the invention have quick, sensitive, intuitive, of low cost, operation letter Just the features such as detection that is, harmless, being easy to great amount of samples, make tobacco grower easy to operate, diagnosis is promptly and accurately.

Importantly, the specificity of the virus of the ELISA test strip of the present invention is very strong, it is specific to Tobacco-growing areas in Guangdong The detection of PVY.The popular strain that the key antibody that the test strips use is specific to Tobacco-growing areas in Guangdong PVY builds to obtain, made Standby targetedly monoclonal antibody sensitivity is very high, can reach 10^-2Mg/mL, the quick inspection to Tobacco-growing areas in Guangdong PVY viruses Survey has great importance.

In addition, the entire small product size of the present invention is small, easy to carry, instrument and equipment is not needed, it is easy to operate.It live can examine It surveys, result can be gone out in 3~10min；As a result it can be judged according to the depth of T line colors by naked eyes, be very suitable for big Batch sample carries out live primary dcreening operation, there is very much application value in actual production, has powerful popularizing application prospect.

Description of the drawings

Fig. 1 is the recombinant vector schematic diagram that CP-P-GD is connected to pET30a-GST carriers.

Fig. 2 is CP-P-GD protein mini-expression detection of expression electrophoretograms；M：Marker, 1：Target protein, 2：Control is not induced.

Fig. 3 is that CP-P-GD albumen great expressions detect electrophoretogram；M：Marker, 1：Supernatant, 2：Precipitation.

Fig. 4 is CP-P-GD protein purification results；M：Marker, 1：Sample dilutes 100 times.

Fig. 5 is the schematic diagram of colloidal gold fast detecting test paper strip.

Fig. 6 is PVY substance virus colloid fast gold test strip detection results；(left side is negative sample, and the right is sun Property sample).

Specific implementation mode

It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art Agent, method and apparatus.

Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.

The separation of 1 Tobacco-growing areas in Guangdong's PVY Strain of embodiment

1,2012~2014 years, from the Nanxiong of Shaoguan City of Guangdong Province, begin to flourish, newborn source, Lechang, the company continent of Qingyuan City, Meizhou 29,9 counties the Ge Di town such as Wuhua, Jiangling, Dabu and the Mei County in city, the tobacco of performance common mosaic is planted in the vega in 33 villages It is sampled in strain.Sample point covers all cigarette districts in Guangdong Province.Totally 72 samples.It is subjected to biology and ELISA respectively Separation, the identification of (enzyme linked immunosorbent assay (ELISA)).

It isolates to obtain 49 PVY isolates, has all obtained ELISA detection verifications.Biology identification the result shows that：It is adopted PVY isolates belong to common strain.

2, to the full CP (coat protein) of PVY viruses) gene expands.By PCR product glue recycle after with pMD- 18T carriers connect, conversion E. coli DH5 α.Picking positive colony expanding propagation culture, alkaline lysis extract plasmid Afterwards, double digestion identification is carried out, the positive transformant containing recombinant plasmid is obtained.Send positive transformant to Beijing AudioCodes biotechnology Co., Ltd is sequenced.

It is 796bp (sequence is as shown in SEQ ID NO.3) that obtained PVY CP genes, which are sequenced, it is respectively designated as CP- P-GD。

Into the database of NCBI, CP-P-GD sequences are compared with the data of lane database respectively with Blast tools Analysis.

3, analysis result is shown：The similitude of the sequence for the PVY CP genes that CP-P-GD sequences are reported with other is 80.5%~95%.This illustrates that this research obtained accounts for the PVY isolates of main advantage status and common PVY in Tobacco-growing areas in Guangdong There is some difference for strain.

Embodiment 2 prepares CP gene prokaryotic differential proteins

1, CP-P-GD prokaryotic expression proteins are prepared, CP-P-GD is building up on pET30a-GST carriers, convert BL21 bacterium Strain carries out prokaryotic expression, purifying expression albumen.

Relevant information is summarized as follows shown in table 1.

Table 1

2, the specific method is as follows.

(1) objective gene sequence：Sequence after being optimized according to e. coli codon, the enzyme enzyme site EcoR I in both ends and Xho I.Target gene is synthesized to pUC57 carriers.

(2) by digestion, connection, target gene is connected to pET30a-GST carriers, the recombinant vector signal after structure Figure is as shown in Fig. 1.

Genetic fragment digestion：43 μ l recombinant plasmids, 1 μ l EcoR I, 1 μ l Xho I, 5 10 × Buffer of μ l, 37 DEG C of mistakes Night reacts.(Ago-Gel DNA QIAquick Gel Extraction Kits, BPI).

Carrier digestion：43 μ l carriers (pET30a-GST) plasmids, 1 μ l EcoR I, 1 μ l Xho I, 5 μ l 10 × Buffer, 37 DEG C of reaction overnights.(Ago-Gel DNA QIAquick Gel Extraction Kits, BPI).

Connection：1 μ l carrier endonuclease bamhis, 3 μ l gene endonuclease bamhis, 1 μ l ligases (BPI), 52 × Rapid of μ l Buffer, mixing react at room temperature 30min.

(3) recombinant vector is converted into BL21 competent cells：

1) the 100 μ l competent cells (BL21) for taking out -80 DEG C of preservations are placed on ice slowly defrosting；

2) competent cell is added in the pipe of 1 μ l recombinant plasmids, mixing places 30min on ice；

3) 42 DEG C of heat shock 90s；

4) after ice bath 2min, the LB culture mediums of 800 μ l non-resistants are added；

5) 37 DEG C of culture 45min；

6) 5000rpm centrifuges 3min, abandons most of supernatant, stays about 100-150 μ l, and thalline is resuspended, and selection has corresponding resistant LB tablets, coated plate；

7) it dries, overnight incubation is inverted in 37 DEG C of incubators.Then sequence verification is correct.

(4) a small amount of detection of expression：

1) it is chosen in monoclonal to 1.5ml LB liquid mediums from the tablet of conversion, 37 DEG C, 200rpm cultures；

2) culture is induced to OD=0.6, IPTG (0.5mM), and 37 DEG C, 200rpm cultivates 2h；

3) bacterium solution for taking 1ml to induce, 12000rpm centrifuge 1min, abandon supernatant, precipitation 50-100 μ l 10mM Tris- HCl (pH8.0) solution dispels and (amount of buffer solution is added depending on biomass)；

4) 2 × loading buffer isometric with buffer solution is added, 100 DEG C are boiled 5min, electrophoresis detection (15%SDS- PAGE)。

As a result as shown in Fig. 2, there is correct band of expression.

(5) great expression：

1) the correct bacterial strain of verification is selected, in the bacterium solution to 5ml LB liquid mediums for connecing 5~10 μ l activation, 37 DEG C, 200rpm, culture；

2) bacterium solution of culture is transferred to 500mL LB liquid mediums to mix, 37 DEG C, 200rpm, culture to OD=0.6, IPTG (0.5mM) induces 4h；

3) bacterium is largely received：With the big concentrator bowls of 400ml, 6000rpm centrifuges 5min, abandons supernatant；

4) carrying out ultrasonic bacteria breaking：Precipitation is dispelled with 25ml 10mM Tris-HCl (pH 8.0) solution, ultrasound；

5) electrophoresis determines expression-form：The bacteria suspension after 100 μ l ultrasounds (500W 90 times, each 3s, is spaced 6s) is taken, 12000rpm centrifuges 10min, takes 50 μ l supernatants to be managed to another EP, after supernatant removal is clean, precipitates with 50 μ l 10mM Tris- HCl (pH 8.0) solution dispels, and 50 μ 2 × loading of l buffer is added, 100 DEG C are boiled 5min, electrophoresis detection (15%SDS- PAGE)。

As a result as shown in Fig. 3, albumen has expression, and main expression is in precipitation.

(6) protein purification (washing inclusion body)：

1) precipitation that ultrasound centrifugation obtains is resuspended in 20~30ml 10mM Tris-HCl (pH8.0) solution, stands 10min；

2) 12000rpm, centrifuges 10min, and supernatant is transferred in another pipe and preserves；

3) precipitation is resuspended in 20~30ml 10mM Tris-HCl (pH8.0) solution, stands 10min；

4) 12000rpm centrifuges 10min, abandons supernatant；

5) repeat 3), 4) it is primary；

6) a small amount of 10mM Tris-HCl (pH8.0) solution is first added, precipitation is resuspended, then add 5~10ml urea containing 8M 10mM Tris-HCl (pH8.0) solution soluble protein；

7) 12000rpm centrifuges 10min, collects supernatant, takes 50 μ l electrophoresis (15%SDS-PAGE).

As a result as shown in Fig. 4, purifying obtains destination protein CP-P-GD.

The hybridoma cell strain of the special monoclonal antibody of the structure expression PVY viruses of embodiment 3

The CP gene prokaryotic differential proteins obtained using embodiment 2, with expression protein immunization mouse；Carry out fusion examination It tests, obtains the positive hybridoma cell strain for generating the special monoclonal antibody of PVY viruses.

1, specific method is as shown in table 2.

Table 2

2, the specific method is as follows：

(1) it is immunized

1) " PVY " is used, by the amount of 60ug albumen/mouse, 4 SPF BALB/c female mices of subcutaneous initial immunity are compiled Number it is：1,2,3,4.

2) just exempt from after two weeks, subcutaneous first time booster immunization, be immunized amount be 30ug albumen/only.

3) first time booster immunization after two weeks, subcutaneous second of booster immunization, be immunized amount be 30ug albumen/only.

4) second of booster immunization after two weeks, subcutaneous third time booster immunization, be immunized amount be 30ug albumen/only.

5) after a week, eye socket takes blood to second of booster immunization, surveys serum titer.

(2) immunizing potency detects：

With " PVY ", 2ug/ml, 4 DEG C of coatings are overnight；2% milk, 37 DEG C of closing 2h；Serum 2 times of gradients since 200 times Dilution, blank control (blank) are PBS, and negative control (negative) is 200 times of dilutions of negative serum.

3 immunizing potency of table (potency is the corresponding dilution of the minimum OD readings more than maximum OD/2)

No. 2 mouse of impact, which are chosen, according to result does cell fusion experiment.Before fusion, with immunogene " PVY " 50ug, abdominal cavity punching Hit immune No. 2 mouse.

(2) cell fusion

Mouse boosting cell and SP2/0 cells are taken, is merged using PEG methods.Cell semisolid culturemedium has been merged (to contain HAT screening and culturing) is carried out.

1) experiment equipment：(including three scissors, three are tweezers, a cell sieve, a syringe for the surgical instrument of sterilizing Inner core, a plate), wet box, 2 500ml beakers, 2 50ml centrifuge tubes, 3 15ml centrifuge tubes.

2) experiment reagent：IMDM culture mediums；IMDM complete mediums (contain 15% serum)；2.2% methylcellulose：Factory Family：SIGMA, article No.：M0262-100G；Newborn bovine serum 10ml；PEG1500：Producer：Roche, article No.：78364；HAT：Factory Family：Sigma, article No.：H0262-10VL；HT：Producer：Sigma, article No.：H0137-10VL.

3) fusion experiment step

A. it is blown and beaten from culture bottle wall by sp2/0 cells in good condition are soft, is drawn into 50ml centrifuge tubes.

B. mouse plucks eyeball and takes blood, then neck is drawn to put to death, is put into 75% alcohol and impregnates 5min.

C. the IMDM that a small amount of serum-free is poured into plate, cell sieve and plunger are put into plate.Use scissors The spleen that mouse is removed with tweezers, is put into cell sieve.Lightly spleen is fully pulverized with the inner core of syringe, it is thin by what is ground Born of the same parents are drawn into the centrifuge tube of dress sp2/0, centrifuge 1500rad/min, 5min.

D. the thymus gland that mouse is removed with scissors and tweezers, pulverizes.By in the thymocyte ground to 15ml centrifuge tubes, then add The HAT for entering 1ml is placed on spare in incubator.

E. the cell that will have been centrifuged, outwells supernatant, and cell is carefully gently blown to even, centrifugation with the IMDM of serum-free (1500rad/min, 5min).

F. the cell conditioned medium centrifuged is outwelled as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37 In DEG C warm water, it is slowly added to the PEG of 1ml in 1 minute, after adding, 1min is stood in warm water.Then it is slowly added in 2min The IMDM of the serum-free of 2ml is then slowly added to the IMDM of 8ml serum-frees in 2min.Centrifuge 1000rad/min, 5min.

G. supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours into the ready thymocyte in front. The sterilized semisolid culturemediums of 25ml are added, are mixed well.Then it uniformly pours into 30 Tissue Culture Dish.Cell is trained Foster ware is put into wet box, is then placed in incubator and is cultivated.

(3) clone is chosen

10 plate × 93 cell monoclonals are chosen, 96 porocyte culture plates is incubated at and (uses thymocyte bed board, 100ul/ in advance Hole).

(4) monoclonal cell 1 sieves

With " PVY " wrapper sheet, ELISA method is used to the clone selected, does and screens for the first time, obtain 19 plants of positive hybridomas Cell strain.

1) experiment reagent：

Coating buffer：Sodium carbonate-bicarbonate buffer solution, pH9.6

PBS buffer solution pH7.4

Confining liquid：2% milk in PBS

Washing lotion：PBS-T (0.05% tween, PBS)

Developing solution:1%A liquid+10%B liquid (A liquid：1%TMB in DMSO；B liquid：0.1%H2O2 in lemon acid bufferings Liquid)

Terminate liquid：2M sulfuric acid

Secondary antibody：Goat anti-mouse IgG/HRP

2) experimental procedure

" PVY " is diluted with coating buffer, final concentration of 2ug/ml, the holes 100ul/, 4 DEG C, overnight；Wash liquid is used afterwards 3 times.

A.2% milk confining liquid is closed, the holes 200ul/, 37 DEG C of incubators, 2h；Wash liquid is used afterwards 3 times.

B. addition primary antibody (cells and supernatant), negative control (SP2/0 culture supernatants), blank control (PBS), the positive are right It is the holes 100ul/, 37 DEG C of incubators, 1h according to (1000 times of dilutions of positive serum PBS)；Wash liquid is used afterwards 3 times.

C. the secondary antibody that PBS dilutes 20000 times, the holes 100ul/, 37 DEG C of incubators, 1h is added；Wash liquid is used after taking-up 3 times.

D. it develops the color, the holes developing solution 100ul/, developing time is 5min or so.

E. 50ul terminate liquids are added per hole to terminate.

F. dual wavelength (450,630) surveys light absorption value, and record preserves data.Include that the hybridization being positive is screened to immune protein Data, positive control reading, blank control reading and the negative control reading of tumor cell strain.

It screens to obtain according to result and the hybridoma cell strain being positive is screened to immune protein, totally 19 plants.

(5) monoclonal cell 2 sieves

By 19 plants of positive cell strains, with " PVY " and label protein, wrapper sheet does second of sieve using ELISA method again Choosing, obtains 5 plants of positive hybridoma cell strains.

(6) screen 5 plants of positive cell strains are subjected to subgroup identification, finally obtain the positive hybridization of 3 plants of IgG types Tumor cell strain.

1) experiment reagent

Coated antibody：(Southern Biotech)

Confining liquid：2%BSA+3% sucrose in PBS；

Developing solution:0.2ml A liquid+10ul 30%H₂O₂In 10ml B liquid (A liquid：15mg/ml ABTS in H2O；B liquid： Citrate buffer solution, pH4.0)

2) various subclass secondary antibody：(Southern Biotech) experimental procedure：

A. it uses 100mM PBS (pH7.4) to dilute coated antibody to 0.5ug/ml, 0.1ml is added per hole, 4 DEG C, is stayed overnight.

B.PBS-T is washed 2 times, and 200ul confining liquids are added per hole, and 370C is incubated 2h.

C.PBS-T is washed 3 times；100ul hybridoma supematants are added per hole, 370C is incubated 1h.

D.PBS-T is washed 3 times；With confining liquid 1：10000 (κ, λ) or 1：The antibody of the diluted HRP labels of 20000 (others) 0.1ml is separately added into per hole in hole appropriate, and 370C is incubated 1h.

E.PBS-T is washed 3 times；Adding 50ul substrate solutions per hole, 10~20min is interior to survey light absorption value in dual wavelength (450,630), Record preserves data.

Table 4

The hybridoma cell strain for finally obtaining the special monoclonal antibody of 19 plants of generation PVY chooses best one plant and cultivates preservation, and It is named as BALB/c-15-8, China Committee for Culture Collection of Microorganisms's common micro-organisms is preserved on June 30th, 2016 Center, deposit number are CGMCC No.12677, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

Embodiment 4 prepares PVY substance virus colloidal gold Rapid detection test strips

1, experiment material

(1) colloid gold particle：Sodium citrate reduction method is prepared (30nm)

(2) specific antibody of PVY viruses

The positive hybridoma cell strain BALB/c-15-8 of embodiment 3 is subjected to culture expression, Protein A is carried out and crosses column Purifying, obtains the specific monoclonal antibody of PVY viruses.

(3) secondary antibody：Goat-anti-Rabbit Ig G

The pH of specific antibody colloid gold label is 8.2, and the pH of secondary antibody is 9.0.

Gold labeling antibody makees stabilizer with BSA.

2, instrument and equipment：

JY-EQ03 continous ways draw film instrument, JY-EQ02 metal spraying machines, and JY-EQ01 cuts formula cutter, I JY-EQ05 pressure shells Machine.

3, consumptive material：

JY-D101 DB-6 bottom plates, JY-X115 H5072 blotting papers, JY-BX101 gold-labelled pads, JY-JZ112 3 sample pads of fusion, JY-C111 A-11 plastic clips.

4, the assembling of test strips

(1) as shown in Fig. 5, by backing (backing), sample pad, water absorption pad (absorbent filter), nitrocellulose filter (NC Film), gold-labelled pad (colloidal gold pad) stick together, the test strips of 4.5mm wide are cut into using cutting machine, in 4 DEG C of kept dries It is spare.

(2) test strips assembling condition：

For the group reload request of test strips under the environment of the constant drying of room temperature, humidity is big or temperature is excessively high can influence glass fibers Dimension, the activity of the property of NC films and secondary antibody, coated antibody, gold labeling antibody, and then influence the Tomography Velocity of test strips and develop the color anti- It answers.When each section assembles, to paste not draw NC films closely, otherwise can influence outlet effect.This test strips 25 DEG C of room temperature, Humidity is assembled under the conditions of being less than 40%.

5, the structure of PVY substance viral diagnosis test strips of the invention is described as follows：

It is made of sample pad, colloidal gold pad, NC films, absorbent filter and backing；The sample pad, colloidal gold pad and NC films are pressed It is incorporated in successively on backing the same face according to sequence from left to right, from top to bottom, the end overlapping of colloidal gold pad and NC films；It is described Absorbent filter is incorporated in the other end of NC films；The PVY specific antibodies of colloid gold label, the NC are coated in the colloidal gold pad Detection line (T lines) and control line (C lines) are provided on film, and detection line (T lines) is located between colloidal gold pad and control line (C lines) Position；The specific antigen of PVY viruses is coated in detection line (T lines)；Gold labeling antibody in the colloidal gold pad can be with inspection Antigen binding on survey line is reacted and is developed the color；It is coated with secondary antibody on control line (C lines).

The detection basic principle of the test strips is as follows：

The PVY specific antibodies of colloid gold label are adsorbed on bonding pad (i.e. colloidal gold pad), the specific antigens of PVY viruses with On ribbon is fixed at the p-wire (T lines) of nitrocellulose membrane, secondary antibody is fixed at the control line (C lines) of nitrocellulose membrane. It after sample to be checked is added in the sample pad of test strips one end, moves forward through capillary action, dissolves the colloid on bonding pad When reacting to each other after gold label specific reagent, then being moved to fixed antigenic domains, the conjugate of object to be checked and golden labelled antibody It specifically binds therewith again.

When sample to be tested contains PVY viruses, the spy of it and the corresponding virus for the colloid gold label being dissolved in sample pad Xenoantibody reacts to each other；When being moved to fixed antigenic domains again, not enough gold labeling antibodies and fixed antigen are anti- It answers, it is the positive not have the appearance of rufous lines, experimental result at T lines.Free gold labeling antibody or gold labeling antibody complex logistics When through at C, there is rufous quality control band with two anti-bindings at this.

When sample to be tested does not have PVY viruses, it does not react with the colloidal gold labeled monoclonal antibody being dissolved on bonding pad； When being moved to fixed antigenic domains again, there are enough gold labeling antibodies and fixed antigen-reactive, and is trapped and is gathered in T lines On, rufous band can be observed by the naked eye, experimental result is feminine gender.

The sample detection of 5 colloidal gold fast detecting test paper strip of embodiment

1, take the blade of the tobacco and health tobacco that have infected PVY viruses as experiment material, each 0.2g adds PBS slow Fliud flushing (pH8.0,0.01M), is ground, and not dilute leaf with tap water.

50uL lapping liquids are respectively taken to be added in the test strips sample pad prepared, sentence read result after five minutes.

2, result is as shown in Fig. 6.There is apparent rufous band in quality inspection band at the test strips C of susceptible tobacco sample, Experimental result is the positive.

The apparent 2 rufous bands of band band appearance are detected at quality inspection and T at the test strips C of health tobacco sample, it is real It is feminine gender to test result.

The results show that the PVY substance virus colloidal gold Rapid detection test strips of the present invention are good to the detection result of PVY viruses It is good.

The sensitivity technique of 6 colloidal gold fast detecting test paper strip of embodiment

1, the 0.1g tobacco diseases sample PBS of 0.01mol/L is diluted into 6 series concentrations, is mixed in equal volume with sample treatment liquid After conjunction, final concentration of 10,1,10^-1、10^-2、10^-3、10^-4Mg/mL, it is negative right to be mixed into equal volume with sample treatment liquid with PBS According to measurement test strips sensitivity.

2, the results show that when a concentration of the 10 of tobacco disease sample^-3When mg/mL, the test strip of test strips is smudgy.Cause This, the detection sensitivity of test strips of the present invention can reach 10^-2mg/mL。

Claims (7)

1. the hybridoma cell strain BALB/c-15-8 of the special monoclonal antibody of one plant of generation PVY, which is characterized in that June 30 in 2016 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center day, deposit number is CGMCC No.12677, Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

2. the special monoclonal antibody of PVY a kind of, which is characterized in that by hybridoma cell strain BALB/c-15-8 described in claim 1 points Secrete generation.

3. application of the special monoclonal antibody of PVY described in claim 2 in terms of preparing PVY viral diagnosis products.

4. application according to claim 3, which is characterized in that the PVY viruses refer to the PVY viruses of Tobacco-growing areas in Guangdong.

5. a kind of PVY substances virus colloidal gold Rapid detection test strip, which is characterized in that by sample pad, colloidal gold pad, NC films, Absorbent filter and backing composition；The sample pad, colloidal gold pad and NC films are tied successively according to sequence from left to right, from top to bottom It closes on backing the same face, the end of colloidal gold pad and NC films is overlapped；The absorbent filter is incorporated in the other end of NC films；It is described It is coated with the PVY specific antibodies of colloid gold label in colloidal gold pad, detection line and control line are provided on the NC films, and detect Position of the line between colloidal gold pad and control line；The specific antigen of PVY viruses is coated in detection line；It is coated on control line There is secondary antibody；The PVY specific antibodies are the special monoclonal antibodies of the PVY described in claim 2.

6. PVY substances virus colloidal gold Rapid detection test strip described in claim 5 is in the application of the context of detection of PVY viruses.

7. application according to claim 6, which is characterized in that the PVY viruses refer to the PVY viruses of Tobacco-growing areas in Guangdong.