CN102174107B - Monoclonal antibody for plague bacillus Pla (plasminogen activator) and application thereof - Google Patents

Abstract

The invention relates to a monoclonal antibody for a plague bacillus Pla (plasminogen activator). The monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of C201091 or C201094 in the CCTCC (China Center for Type Culture Collection). The invention also relates to a preparation method of the monoclonal antibody and the hybridoma cell strain for generating the monoclonal antibody. The invention further relates to a piece of monoclonal antibody colloida gold immuno-chromatographic test paper and application thereof to plague bacillus detection and/or auxiliary plague diagnosis.

Description

Monoclonal antibody and the application thereof of plague bacillus plasminogen activating factors Pla

Technical field

The present invention relates to the monoclonal antibody of plague bacillus plasminogen activating factors Pla, the application in detecting Pla of the hybridoma cell strain that relates to described MONOCLONAL ANTIBODIES SPECIFIC FOR method simultaneously and produce described monoclonal antibody, described monoclonal antibody and colloidal gold immuno-chromatography test paper strip and the application in detecting plague bacillus and/or the auxiliary diagnosis plague thereof that contains described monoclonal antibody.

Background technology

The plague (Plague) is the natural epidemic disease source property deadly infectious disease that is caused by Yersinia (Yersinia pestis).Plague immunodetection is an important step in the plague preventing and controlling, brings into play crucial effects in its discovery in the plague, epidemic-stricken area processing and the clinical diagnosis.

Traditional plague immunodetection, main line are at F1 antigen.F1 antigen is a kind of protein clostridium by cafl genes encoding on the Yersinia pMTl plasmid.For a long time, F1 antigen is considered to the specific antigens of plague bacillus, is considered to the more believable plague based on antigen, the antibody detection method of F1 and detects and diagnostic method.Yet nature has found to exist the plague bacterial strain of natural disappearance F1 antigen, and in addition, F1 antigen is a kind of temperature adjusting antigen expressed, great expression in the time of 37 ℃, and in the time of 28 ℃, will not express or trace expression, this has also limited its applied research scope.Detection and the diagnostic techniques of development except F1 antigen is comparatively necessary, is an important topic in the plague preventing and controlling.

Plague bacillus plasminogen activating factors (Plasminogen activator, Pla) be the plasmid-encoded cell surface protein enzyme of the distinctive pPCPl of plague bacillus, studies show that, plague bacillus plasminogen activating factors Pla albumen is plague bacillus is diffused into the recycle system from subcutaneous infection important virulence factor (Sodeinde OA, Subrahmanyam YV, Stark K, et a1.A surface protease and the invasive character of plague[J] Science, 1992,258 (6): 1004-1007; Goguen JD, Hoe NP, Subrahmanyam YVBK.Proteases and bacterial virulence:a view fromthe trenches[J] .Infect Agents Dis, 1995,4 (1): 47-54), in the plague bacillus pathogenic course, play a significant role.And the transcribing of Pla, translate and be not subjected to temperature limitation.Detect at Pla albumen, for detection and/or the auxiliary diagnosis plague of plague bacillus, particularly significant for the plague bacterial strain (the particularly detection of the communication media flea of the plague) of F1 antigenic deletion plague bacterial strain and non-37 ℃ of conditions growth.

At present the detection to plague bacillus Pla mainly is to adopt round pcr, and has obtained and detect effect preferably.Yet this method requires than higher technological operation, and testing process needs a few hours, and the time is longer.

On the other hand, monoclonal antibody is compared to polyclonal antibody, have high specificity, advantage that susceptibility is high, can be used as the important tool of the biological function research of related antigen, be used for experiments such as Western blot, immunoprecipitation, immunocytochemistry, significant for the detection of the research of antigen, relative disease, diagnosis, prognosis judgement etc.

But do not see at present the report of domestic monoclonal antibody relevant for plague bacillus plasminogen activating factors Pla.

Summary of the invention

One object of the present invention is to provide the monoclonal antibody of plague bacillus plasminogen activating factors Pla, and this monoclonal antibody has specificity to plague bacillus plasminogen activating factors Pla albumen.

Another object of the present invention is to provide the hybridoma cell strain that produces described monoclonal antibody.

Another object of the present invention is to provide preparation described monoclonal antibody method.

Another object of the present invention is to provide the application of described monoclonal antibody in detecting Pla albumen.

Another object of the present invention is to provide a kind of colloidal gold immuno-chromatography test paper strip of the Pla of detection albumen, and it has the plague bacillus of detection and/or the effect of the auxiliary diagnosis plague.Contain monoclonal antibody of the present invention on this colloidal gold immuno-chromatography test paper strip.

The present invention uses the monoclonal antibody that hybridoma technology has prepared plague bacillus plasminogen activating factors Pla.

At first, one aspect of the present invention has provided and a kind of plague bacillus plasminogen activating factors Pla albumen has been had specific monoclonal antibody." specificity of antibody " described in the present invention refers to that monoclonal antibody is to the recognition capability of corresponding antigens or approximate antigenic substance.The specificity height, the ability of identification antigen is just strong.In a specific embodiments of the present invention, monoclonal antibody of the present invention has specificity to GST (glutathione s transferase)-Pla fusion rotein, natural Pla albumen, and glutathione s transferase is not had specificity.In a preferred specific embodiments of the present invention, described monoclonal antibody is to be the hybridoma cell strain secretion generation of CCTCC NO.C201091 or CCTCC NO.C201094 by preserving number.The antibody type belongs to IgG1 and IgG2a respectively.

Another aspect of the present invention provides the hybridoma cell strain that produces described monoclonal antibody.Regulation according to " Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure ", the two strain of hybridoma strains that produce monoclonal antibody of the present invention have been deposited in one of international depositary institution---Chinese typical culture collection center (China Center for Type Culture Collection, CCTCC, address: Chinese Wuhan City Wuhan University, postcode: 430072), preservation date: on September 16th, 2010, deposit number: CCTCC NO.C201091, CCTCC NO.C201094.

Another aspect of the present invention provides a kind of preparation described monoclonal antibody method.

In embodiments of the invention, be that Pla albumen and known label are formed recombinant protein as antigen, to prepare monoclonal antibody of the present invention.Described label is selected from glutathione-S-transferase label (GST label), in a specific embodiments of the present invention, and the recombinant protein that adopts GST and the Pla albumen of removing signal peptide to form.

Preparing the used antigen of monoclonal antibody of the present invention can be by ordinary method preparation well known by persons skilled in the art.For example Pla albumen of the present invention can be recombinant protein, or extracts from plague bacillus and natural Pla albumen that purifying obtains.

In a preferred specific embodiments of the present invention, as the antigen immune mouse, get the splenocyte of mouse as the plasmocyte (immunocyte) that can produce antibody with the GST-Pla fusion rotein, merge with the myeloma cell of syngeneic animal.Screening can produce the hybridoma of purpose antibody, carries out cloning and cultivates, and set up the hybrid cell strain.Aforesaid method only is exemplary, for example can use the same method the immunity other Mammalss, with its splenocyte as immunocyte.Can also select the myeloma cell that suits to be used for merging, for example be used for myeloma cell from rat, mouse or hamster.

Immunocyte and myeloma cell's fusion can carry out according to ordinary method (Milstein et al., Mechods Enzymol., 1981,73,3-46).More particularly, immunocyte and myeloma cell can fully mix in substratum according to 1: 4～1: 10 ratio, carry out cytogamy under the effect of merging promotor, PEG (molecular weight 1000～6000) solution that for example will be preheated to 37 ℃ adds substratum with the ratio of 30%～60% (W/V).In addition, can also suitably add adjuvant (as dimethyl sulfoxide (DMSO)) as required to strengthen syncretizing effect.The substratum that be used for to merge comprises that RPMI-1640 substratum, DMEM substratum and other can be used for the substratum of this type of cultivation, and adds an amount of materials such as newborn calf serum as required.

Can be with common selection substratum screening hybridoma, for example with the HAT substratum that contains xanthoglobulin, aminopterin, thymidine.In the HAT substratum, continue to cultivate time enough, so that cell (non-fused cell) death beyond the hybridoma is generally several days to several weeks.

Screening produces the hybridoma of purpose antibody and carry out mono-clonalization then.The hybridoma of the generation monoclonal antibody of the present invention that obtains can go down to posterity in ordinary culture medium and cultivate or preserve for a long time in liquid nitrogen.

When preparing monoclonal antibody of the present invention from hybridoma, can from hybridoma vitro culture supernatant liquor, obtain antibody, perhaps hybridoma be injected suitable Mammals and obtain antibody from animal ascites.Preceding a kind of method is suitable for obtaining highly purified antibody, and a kind of method in back is suitable for obtaining in a large number, high-titer antibody.By the antibody that aforesaid method obtains, can use the ordinary method purifying, for example saltout, methods such as gel-filtration, affinity chromatography.

In a specific embodiments of the present invention, described preparation monoclonal antibody method comprises with GST-Pla fusion protein immunization mouse, the splenocyte and the myeloma cell that get mouse are merged, filter out and to secrete natural responding property of Pla albumen, glutathione s transferase is not had the hybridoma of reactive monoclonal antibody, from hybridoma supernatant or the ascites that produces of the animal behind the injection hybridoma, obtain monoclonal antibody.In this scheme, specifically be the GST-Pla fusion rotein routine immunization BALB/c mouse with purifying, serum titer reaches 1: 10 ⁴～10 ⁵The time, immune mouse spleen cell and Sp2/0 myeloma cell are merged.HAT selects to cultivate the 10th day, and the hybridoma fusion rate is more than 65%.Merge the back and carried out the ELISA screening first time, subclone on the 10th day, after this repeat ELISA screening and subclone 3～4 times, until clone's positive rate 100%.3 strain of hybridoma strains have been filtered out in this scheme, respectively called after hybridoma cell strain 15B ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄, the monoclonal antibody of the anti-Pla of this 3 strain of hybridoma strain energy stably excreting.With these strain of hybridoma enlarged culturing and abdominal injection BALB/c mouse, the energy inducing mouse produces the ascites of anti-Pla, and ascites output 2～10ml/, it is tired and reaches 1: 10 ⁶This has created better condition for being applied to diagnostic techniques.

Hybridoma cell strain 15B ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄The monoclonal antibody of secreted generation (called after monoclonal antibody 15B among the present invention ₈, monoclonal antibody 19A ₄, monoclonal antibody 14H ₄), have antigen-binding specificity highly.In a specific embodiments of the present invention, for analyzing the specificity of Pla monoclonal antibody, the present invention carries out indirect ELISA with the GST of purifying and GST-Pla fusion rotein and natural Pla albumen and detects, experimental result shows, 3 strain monoclonal antibodies are strong positive reaction with purifying GST-Pla fusion rotein and the natural Pla albumen of bag quilt, are negative reaction with the GST albumen that wraps quilt.Be antigen with natural Pla albumen, 3 strain monoclonal antibodies are primary antibodie, and HRP-SPA two anti-ly carries out Western blot experiment, and the result shows that 3 strain monoclonal anti physical efficiencys identify the Pla albumen that extracts in the native protein specifically.

The present invention also provides the purposes of monoclonal antibody of the present invention.

In one embodiment of the invention, the invention provides the application of described monoclonal antibody in the level that detects Pla albumen.The present invention also provides a kind of preparation of the Pla of detection albumen, and said preparation contains monoclonal antibody of the present invention.Monoclonal antibody high specificity of the present invention, purity height, the height of tiring can be applied to the ELISA method simultaneously, Western Blot hybridization, and immunohistochemistry and cellular immunization chemical process detect the expression level of Pla albumen.

Another aspect of the present invention also provides the application of described monoclonal antibody in the colloidal gold immuno-chromatography test paper strip of preparation detection Pla albumen.The invention provides a kind of colloidal gold immuno-chromatography test paper strip of the Pla of detection albumen, contain monoclonal antibody of the present invention on this colloidal gold immuno-chromatography test paper strip.This colloidal gold immuno-chromatography test paper strip that contains monoclonal antibody of the present invention can be used for detecting plague bacillus and/or the auxiliary diagnosis plague.

Because monoclonal antibody of the present invention has the antigen-binding specificity of height, utilize monoclonal antibody of the present invention to set up detection technique, can be used as the detection of plague bacillus and/or the auxiliary diagnosis of the plague, especially significant for the diagnosis of the plague bacterial strain of F1 antigenic deletion plague bacterial strain and the growth of non-37 ℃ of conditions.Therefore, another aspect of the present invention also provides monoclonal antibody of the present invention for the preparation of the application in the preparation that detects plague bacillus and/or the auxiliary diagnosis plague.

The present invention confirms by a large amount of experimental studies: monoclonal antibody of the present invention, antigen-binding specificity with height, main experimental evidence comprises: immune mouse is used the GST-Pla fusion rotein, and the monoclonal antibody that obtains detects confirmation through the ELISA method, the GST-Pla fusion rotein and the natural Pla albumen that are purified with bag are strong positive reaction, and be negative reaction with bag by GST albumen, and antibody titer reaches 10 ^-6Simultaneously, be antigen with natural Pla albumen, 3 strain monoclonal antibodies are primary antibodie, and HRP-SPA two anti-ly carries out Western blot experiment, the result shows that 3 strain monoclonal anti physical efficiencys identify the Pla composition that extracts in the native protein specifically, and does not react with other composition.So, the prepared Pla monoclonal antibody of the present invention is laid a good foundation for function and the mechanism of causing a disease thereof of further furtheing investigate Pla, the function that is conducive to deep discussion Pla, use it for the detection at Pla albumen, for detection and/or the auxiliary diagnosis of plague bacillus, particularly significant for the diagnosis of the plague bacterial strain of F1 antigenic deletion plague bacterial strain and the growth of non-37 ℃ of conditions.

Description of drawings

Fig. 1 shows the SDS-PAGE analytical results of transformant BL21-rPla abduction delivering product P la.Among the figure, 1: molecular weight standard; 2: the cellular lysate postprecipitation behind the transformant BL21-rPla abduction delivering; 3: supernatant behind the cellular lysate behind the transformant BL21-rPla abduction delivering; 4: transformant BL21-rPla induces preceding whole bacterial protein; 5: the product of cellular lysate postprecipitation behind preliminary purification behind the transformant BL21-rPla abduction delivering.

Fig. 2 is hybridoma cell strain 15B of the present invention for the colchicine method checks ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄Chromosome karyotype analysis figure (Giemsa dyeing, * 1000).

Fig. 3 is the SDS-PAGE figure of purifying ascites.Among the figure, M: molecular weight standard; 1:15B ₈A; 2:15B ₈B; 3:19A ₄A; 4:19A ₄B; 5:14H ₄A; 6:14H ₄C; 7:14H ₄D.

Fig. 4 shows that Western blot identifies the experimental result of the natural Pla albumen of described monoclonal antibody specific recognition.Among the figure, M: dye marker in advance; 1:15B ₈2:19A ₄3:14H ₄

Fig. 5 shows the subclass test experience result of described monoclonal antibody.

Fig. 6 A shows that detecting Pla antigen colloidal gold immuno-chromatographic test paper strip detects 28 ℃ of cultivation EV76 thalline experimental results; Fig. 6 B shows that detecting F1 antigen colloidal gold immuno-chromatographic test paper strip detects 28 ℃ of cultivation EV76 thalline experimental results.The microorganism that is used for patented procedure preserves:

(1) secretion monoclonal antibody 15B of the present invention ₈Hybridoma cell strain 15B ₈

Preservation date: on September 16th, 2010;

Depositary institution: Chinese typical culture collection center (CCTCC);

Deposit number: CCTCC NO:C201091;

Classification name: hybridoma cell strain 15B ₈

(2) secretion monoclonal antibody 19A of the present invention ₄Hybridoma cell strain 19A ₄

Preservation date: on September 16th, 2010;

Depositary institution: Chinese typical culture collection center (CCTCC);

Deposit number: CCTCC NO:C201094;

Classification name: hybridoma cell strain 19A ₄

Embodiment

In order more to be expressly understood essence of the present invention, further describe the present invention below by specific embodiment and conjunction with figs., but therefore the present invention is not subjected to any restriction.The experimental technique of unreceipted actual conditions in the following example, usually (third edition [U.S.] Sa nurse Brookers in 2002 etc. are outstanding as " molecular cloning experiment guide " according to normal condition, Science Press) condition described in, or the condition of advising according to manufacturer.

Below among each embodiment, all commercially available acquisitions of used original experiment material, instrument apparatus and reagent below provide the source of main experiment material, instrument apparatus and reagent:

1 experiment material

1) BALB/c mouse: available from Shanghai Experimental Animal Center (conformity certification SCXK (Shanghai) 2007-0005),

Unming Medical College's Experimental Animal Center (conformity certification SCXK (Yunnan) 2005-0008).

2) mouse myeloma cell line Sp2/0: available from cloud mcroorganism Science and Technology Ltd..

3) rPla albumen: available from Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an.

4) Yersinia pestis EV76 bacterial strain: available from Yunnan Province's prevention and cure of endemic diseases institute strain library.

2 laboratory apparatuss and apparatus

Instrument apparatus title	Producer
		0.22 the disposable filter membrane of μ m	Millipore
0.45 the disposable filter membrane of μ m	Millipore
		6 holes 24 holes 96 porocyte culture plates	Falcon
The cell cryopreservation pipe	Falcon
		96 hole enzyme reaction plates	Coster
Cell counting count board	Sigma
		Milli-Q ultrapure water system	Millipore
PH meter	Orion
		The full-automatic high-pressure steam sterilizer	Hirayama
Bechtop (HF Safe 1200)	Forma
		CO 2Cell culture incubator	Forma
Electronic balance	Sartorius

Inverted microscope	LEICA
		Ordinary optical microscope	Nikon
Liquid nitrogen container	Forma
		General refrigerator	Qingdao Haier
Ultralow Temperature Freezer	Revco ULT-1386-3V
		Wash the plate machine	BIO-RAD 1575
Microplate reader	The BIO-RAD550 type
		Low speed centrifuge	Beijing Medical Centrifugal Machine Factory
AKTA explorer 100，AKTA primer	Amersham Pharmacia
		The acrylamide gel electrophoresis system	BIO-RAD
The film system is changeed in the electricity transfer printing	BIO-RAD Trans Blot SD

3 main agents

Reagent name	Producer
		DMSO (dimethyl sulfoxide (DMSO))	Sigma
HT(50×HT Media Supplement)	Sigma
		HAT(50×HAT Media Supplement)	Sigma
BSA (bovine serum albumin)	Hyclone
		FBS (foetal calf serum)	Hyclone
FCS (new-born calf serum)	Hyclone
		Penicillin, Streptomycin sulphate	Homemade
PEG-1500	Fluka
		Antibody subgroup identification reagent	HyCult Biotechnology
TMB (3,3 ', 5,5 '-tetramethyl benzidine)	BBI
		The HRP-sheep anti-mouse igg	Sigma
Trypan blue	The worker is given birth in Shanghai
		Tween-20	The worker is given birth in Shanghai
Giemsa	The worker is given birth in Shanghai
		EDTA	The worker is given birth in Shanghai
SDS	Invirtogen
		N,O-Diacetylmuramidase	The worker is given birth in Shanghai
Ammonium persulphate, glycine	The worker is given birth in Shanghai
		Xylene Brilliant Cyanine G R-250	The worker is given birth in Shanghai
Methyl alcohol	The worker is given birth in Shanghai
		Glacial acetic acid	The worker is given birth in Shanghai

The preparation of 4 main agents

4.1 cell cultures solution

1) the incomplete substratum of DEME (1000ml): with pure water dissolving DMEM dry powder 13.4g, Na ₂CO ₃3.7g, treat to dissolve fully the back and add penicillin, each 200,000 unit of Streptomycin sulphate, be settled to 1000ml, through 0.45 μ m and 0.22 μ m membrane filtration degerming, packing, 4 ℃ of preservations are standby.

2) DEME perfect medium (1000ml): under the aseptic condition, get 1) the incomplete substratum 800ml of DEME of method preparation adds 200ml FBS/FCS, the jog mixing.

3) trypan blue mother liquor: take by weighing trypan blue 4.0g, thoroughly grind with pure water, filter and remove impurity, room temperature preservation.Be working fluid with 1.8% physiological saline and mother liquor by 1: 1 mixed when using, working fluid mixes with cell cultures suspension equal-volume and carries out viable count.

4.2ELISA experiment solution

1) bag is cushioned liquid (pH 9.6 0.05mol/L carbonate buffer solutions)

Na ₂CO ₃1.59 gram

NaHCO ₃2.93 gram

Adding distil water transfers to 9.6,4 ℃ of preservations to 1000ml with the pH value.

2) lavation buffer solution PBS-T (pH7.4 0.15M)

KH ₂PO ₄0.2 gram

Na ₂HPO ₄12H ₂O 2.9 grams

NaCl 8.0 grams

KCl 0.2 gram

Tween-20 0.5ml

Adding distil water transfers to 7.4,4 ℃ of preservations to 1000ml with the pH value.

3) confining liquid

Bovine serum albumin (BSA) 1.0 grams

Add lavation buffer solution to 100ml;

Or be made into 5～10% with bovine serum and washings and use 4 ℃ of preservations.

4) substrate solution A

0.1M citric acid (19.2g/L) 24.3ml

0.2M Na ₂HPO ₄(28.4g/L) 25.7ml

Adding distil water is to 50ml.The pH value is transferred to 9.6,4 ℃ of preservations.

5) substrate solution B

Citric acid 10.52g

EDTA 1.86g

Glycerine 50ml

Adding distil water transfers to 9.6,4 ℃ of preservations to 2000ml with the pH value.

6) TMB working fluid

DMSO 2ml

TMB 5mg

4 ℃ of preservations.

7) stop buffer

2mol/L H ₂SO ₄, room temperature preservation.

4.3 nucleus type analysis solution

1) colchicine mother liquor

Colchicine 1.0mg

Physiological saline 50ml

4 ℃ of preservations.

2) PBS (1000ml, Giemsa dye liquor damping fluid)

KH ₂PO ₄1.82 gram

Na ₂HPO ₄12H ₂O 19.10 grams

The pH value is transferred to 6.8,4 ℃ of preservations.

3) Giemsa dye liquor mother liquor

Giemsa 0.8g

Methyl alcohol 50ml

Glycerine 50ml

Grind the Giemsa powder in glycerine, 56 ℃ of water bath with thermostatic control 90min add methyl alcohol again, fully stir, and filter in the rearmounted brown bottle and preserve, and dilute 10 times with PBS during use.

4) hypotonic treatment solution (100ml) takes by weighing KCl 0.559g, and adding distil water is to 100ml, room temperature preservation.

4.4SDS-PAGE electrophoresis reagent:

1) 30% acrylamide soln (100ml)

Acrylamide 29.0g

N, N '-methylene-bisacrylamide 1.0g

Need during preparation in the 60ml deionized water, to add above reagent, be stirred well to thorough dissolving, be settled to 100ml again, 4 ℃ of preservations.Because acrylamide is strong neurotoxin, will avoid air-flow during preparation, carries out sfgd. simultaneously.

2) 1.5mol/L Tris-Cl solution (pH 8.8 100ml)

18.2g Tris is dissolved in fully dissolving in the 80ml deionized water, adjusts pH value to 8.8 with concentrated hydrochloric acid, be settled to 100ml again, 4 ℃ of preservations.

3) 1.0mol/L Tris-Cl solution (pH 6.8 100ml)

12.1g Tris is dissolved in fully dissolving in the 80ml deionized water, adjusts pH value to 6.8 with concentrated hydrochloric acid, be settled to 100ml again, 4 ℃ of preservations.

4) 10%SDS solution (pH 6.8 100ml)

10g SDS is dissolved in the 80ml deionized water, and fully stirring and dissolving is adjusted pH value to 6.8 with concentrated hydrochloric acid, is settled to 100ml again, room temperature preservation.Need determine whether before using has SDS to separate out because room temperature is low excessively.

5) 10% ammonium persulfate solution (1.0ml) (Ap)

The 0.1g ammonium persulphate is dissolved in the 1.0ml deionized water, contains 4 ℃ of preservations in brown bottle.Can store 10 days at most, expired need are prepared again.

6) concentrate glue (10%)

30% acrylamide 3.35ml

10％SDS 25ul

0.5mol/L Tris-C1 solution (pH 6.8) 6.25ml

TEMED 3ul

10％Ap 25ul

Ultrapure water 1.5ml

7) separation gel (4%)

30% acrylamide 2.5ml

10％SDS 0.06ml

1.5mol/L Tris-Cl solution (pH 8.8) 1.5ml

TEMED 2ul

10％Ap 30ul

Ultrapure water 1.92ml

8) 5 * SDS-PAGE electrode buffer (storage liquid, 1000ml)

Tris 15.1g

Glycine 94.0g

SDS 5.0g

Above agent dissolves in the 800ml deionized water, is stirred to abundant dissolving, is settled to room temperature preservation behind the 1000ml.The electrophoresis working fluid is 1 * SDS-PAGE electrophoretic buffer, carries out 5 times of dilutions during use and gets final product.

9)5×SDS-PAGE Loading Buffer(5.0ml)

1M Tris-Cl(pH 6.8) 1.25ml

SDS 0.5g

Tetrabromophenol sulfonphthalein 30.0mg

Glycerine 2.5ml

2-ME 0.25ml

In the 10ml test tube, mix mentioned reagent, with the abundant mixing of vibrator, room temperature preservation.

10) coomassie brilliant blue staining liquid (1000ml)

Xylene Brilliant Cyanine G R-250 1.0g

Methyl alcohol 250ml

Glacial acetic acid 100ml

To be settled to 1000ml after the above reagent mix, room temperature preservation.

11) destainer (1000ml)

Glacial acetic acid 100ml

Methyl alcohol 250ml

Deionized water 850ml

4.5Western blot experiment reagent

Electrophoresis reagent is same as above.

1) 10 * commentaries on classics film damping fluid (100ml)

Tris-base 30.3g

Glycine 144g

Methyl alcohol 200ml

Dilution in preceding 1: 10 is used in 4 ℃ of preservations.

2)TBS-T(1000ml)

1mol/L Tris-HCl(pH 7.4) 10.0ml

4mol/L NaCl 37.5ml

50％Tween 20 1.0ml

Room temperature preservation.

3) colour developing liquid is pressed DAB 6mg, H ₂O 2ul, PBS (0.01M, pH 7.4) 10ml preparation.

4) confining liquid

Add the 5.0g skim-milk in 100ml TBS-T washings, stirring makes it to dissolve fully gently.

MONOCLONAL ANTIBODIES SPECIFIC FOR, purifying and the evaluation of embodiment 1 plague bacillus plasminogen activating factors Pla

1 antigen

Reorganization Pla albumen: available from CDC transmissible disease institute; it is the method preparation according to the prior art record; according to Yersinia pestis (Yersinia pestis) Pla gene order (NCBI:GeneID:1149148); and remove its signal coding sequence (the 1st～63 Nucleotide) and terminator codon (the 934th～936 Nucleotide TGA); adopted primer Pla-s and 3 ' end antisense primer Pla-a are rectified in design 5 '; 5 ' end at primer manually adds restriction enzyme site sequence and protection base; be specially Pla-s:5 ' CCG GAATTCTCATCTCAGTTAATACCAAAT-3 ' (SEQ IDNo.:1, the underscore partial sequence is the EcoRI restriction enzyme site), Pla-a:5 ' ATTT GCGGCCGCGAAGCGATATTGCAGAC-3 ' (SEQ ID No.:2, the underscore partial sequence is the NotI restriction enzyme site), it is synthetic that primer entrusts Shanghai to give birth to the worker.The Pla gene fragment that to remove signal coding sequence is connected with the NotI double enzyme site by EcoRI with plasmid pGEX4t-1, recombinant plasmid (Pla-pGEX4t-1) is transformed among the E.coli BL21 (DE3) carries out abduction delivering, the BL21 bacterium that contains recombinant plasmid Pla-pGEX4t-1, produced the rPla fusion rotein that molecular weight is about 58.6kD through inducing, expressed inclusion body products (rPla) and carry out purifying through repetitive scrubbing.Sample is analyzed through SDS-PAGE, and experimental result adopts TotalLab 1.0 software analysis, and its purity is about 85% (the SDS-PAGE analytical results is referring to Fig. 1).

Natural Pla albumen: extract preparation according to ordinary method, concrete operations are as follows: plague EV76 bacterium is inoculated in the LB substratum, hatches 48h for 28 ℃, wash thalline twice with PBS, add 4 times of volumes-40 acetone of low temperature ℃ in advance, abundant mixing, put-40 ℃ spend the night after, 4000r/min, 10min is centrifugal, abandons supernatant, uses precooling washing with acetone thalline 3 times again, abandon most acetone, 37 ℃ of incubators spend the night and make the bacterium powder.The bacterium powder is through ultrasonication, 0～10% ammonium sulfate precipitation, 8000r/min, and 30min is centrifugal, collecting precipitation.

Reorganization GST albumen: available from cloud mcroorganism Science and Technology Ltd..

The foundation of 2 indirect ELISA detection methods

The foundation of indirect ELISA detection method is carried out according to a conventional method, and with rPla albumen, natural Pla albumen and reorganization GST difference coated elisa plate, HRP-sheep anti mouse (available from Sigma) is ELIAS secondary antibody.Adopt the square formation volumetry to determine best antigen coated amount and ELIAS secondary antibody working concentration.According to experimental result, the bag of 3 kinds of antigens is respectively by concentration: natural Pla albumen 5 μ g/ml, reorganization pla protein 10 μ g/ml, GST 2 μ g/ml; The suitableeest working concentration of ELIAS secondary antibody 1: 8000.The concrete operations step is as follows:

(1) bag quilt: after being cushioned liquid known antigens is diluted with bag, every hole adds 0.1ml, and 4 ℃ are spent the night.Washing next day.

(2) sealing: every hole adds 0.2ml 1% bovine serum albumin or 5～10% bovine serums seal., 4 ℃ are spent the night, wash plate.

(3) application of sample: add through the testing sample 0.1ml of certain dilution in the above-mentioned reacting hole that has wrapped quilt, put 37 ℃ and hatched 1 hour, washing.(doing blank well simultaneously, negative control hole and positive control hole).

(4) add enzyme labelled antibody: in each reacting hole, add the enzyme mark second antibody 0.1ml of suitable working concentration, hatch 45～60min for 37 ℃, washing.

(5) add the substrate solution colour developing: in each reacting hole, add the tmb substrate solution 0.1ml that faces the time spent preparation, 37 ℃ of 10～30min.

(6) termination reaction: in each reacting hole, add stop buffer 0.05ml.

The result judges: detect A at enzyme mark detector ₄₅₀Value, which kind of envelope antigen no matter is with positive greater than 2.1 times of negative control value.Detect with rPla and natural Pla and all positively just can confirm as positive hybridoma.

The immunity of 3BALB/c mouse

Select 16 of 4～6 ages in week, physique amount 16～20g, female BALB/c mouse, be divided into 4 groups (A～D group) at random, 4 every group carry out immunity (immunizing antigen be aforementioned available from the CDC transmissible disease reorganization Pla albumen).Carry out 4 immunity altogether, adopt the subcutaneous multi-point injection in mouse back and abdominal injection, initial immunity is fully emulsified with Freund's complete adjuvant and equivalent antigen, the full adjuvant that toos many or too much for use later on, and be 3 weeks per twice immune pitch time.The immunity back was adopted the tail blood examination in 10～14 days and is surveyed serum antibody titer.Detected result shows that two groups of mice serums of A, B are tired and has all reached 1: 10 ⁴～10 ⁵, can be used for carrying out cytogamy.

Select the high mouse of antibody titer, carried out booster immunization in preceding 3 days in fusion, immunizing dose is every mouse 200 μ g, abdominal injection.The fundamental immunity situation sees the following form:

The foundation of 4 hybridoma cell strains

4.1 merge precellular preparation

1) myeloma cell's preparation

Merge preceding 1 week, get the Sp2/0 myeloma cell who preserves in the liquid nitrogen, 37 ℃ of water-baths are thawed rapidly, and with DEME nutrient solution suspension cell, the centrifugal 10min of 1000r/min abandons supernatant.To contain 20% bovine serum DEME nutrient solution re-suspended cell, put 5%CO ₂, cultivate in 37 ℃ of incubators.When cell grows to logarithmic phase, by the cultivation of going down to posterity of 1: 3～1: 10 dilution proportion.Merge and adjusted cell density in preceding 2～3 days, it is vigorous, in good condition to make it be in growth when merging.The same day of merging, the myeloma cell is blown down, make suspension with the centrifugal 10min of 1000r/min, abandon supernatant, the incomplete nutrient solution re-suspended cell with an amount of carries out cell counting.

2) preparation of feeder cell

Merge the day before yesterday or when cloning again, get BALB/c mouse and take off neck and put to death, 75% alcohol-pickled, disinfect.Cut off skin of abdomen under the aseptic condition, the incomplete nutrient solution of 4～5ml is injected in the mouse peritoneal, massage gently, extract nutrient solution out with syringe again, repeat 2～3 times.The centrifugal 10min of cell suspension 1000r/min that obtains abandons supernatant, with DEME complete culture solution re-suspended cell, adjusts cell density, it is dripped 96 porocyte culture plate or culturing bottles, 37 ℃, 5%CO ₂Use after the incubator overnight incubation.

3) preparation of immune mouse spleen cell

Carry out the same day of cytogamy, the BALB/c mouse of booster immunization is got take off neck behind the blood and put to death the about 10min of 75% alcohol-pickled sterilization.Cut off skin, peritonaeum under the aseptic condition, take out spleen, place in the plate that fills the incomplete nutrient solution of an amount of DMEM, reject fat and reticular tissue etc., in stainless steel mesh, push spleen with piston, splenocyte is entered in the nutrient solution, and collecting cell suspension, the centrifugal 10min of 1000r/min abandon supernatant.The full nutrient solution re-suspended cell that toos many or too much for use is counted, and is standby.

4.2 cytogamy and screening

Behind the booster immunization 3 days, aseptic operation gets mouse boosting cell and the Sp2/0 cell merges.Concrete operations:

1) myeloma cell that will count and immune mouse spleen cell place the taper centrifuge tube by 1: 4～10 mixed, and the centrifugal 10min of 1200r/min abandons supernatant, shakes centrifuge tube gently, and cell is uniformly dispersed, and puts 37 ℃ of water bath heat preservations.

2) limit is stirred the cell limit and is added the pre-temperature of 1.0ml to 37 ℃ 50%PEG-1500 solution, drips in the 1min.

3) drip the incomplete nutrient solution of 10ml DMEM gently, add in the 3min, 1000r/min, 10min are centrifugal, abandon supernatant.

4) shake centrifuge tube gently, cell is uniformly dispersed, add 40ml HAT-DMEM-20%FBS nutrient solution, suspension cell is added dropwise to the 96 porocyte culture plates that proxima luce (prox. luc) has prepared feeder cell, every hole 0.1ml with it.It is full that nutrient solution to eighty per cant is added in every hole.

5) culture plate is put 5%CO ₂, cultivate in 37 ℃ of incubators.

6) examine and record the fused cell growing state every day, the HAT complete culture solution is once partly measured in replacing in per 3 days, and cultured continuously 7～14 days is replaced by the HT-DMEM-20%FBS complete culture solution.

7) treat that cell grows to 1/2～1/3 at the bottom of the hole, get cells and supernatant, adopt indirect elisa method to carry out antibody titer and detect, the screening positive clone cell strain, choosing has cross reaction, reorganization GST is not had the cell strain of combination rPla and natural Pla albumen.

In the present embodiment, four mouse (being numbered A2, A4, B2, B4) of having selected Serum Antibody Detection result to stablize, tire high have carried out 4 cytogamy experiments, each time experiment parameter such as following table:

When the selectivity in the HAT-DMEM-20%FBS nutrient solution of the cell after the fusion was cultivated, the cell that only has splenocyte and Sp2/0-myeloma cell to merge in the nutrient solution could be survived, not cell natural death after 5～7 days of Rong Heing.At the bottom of the hole is grown in about about 10 days, cell after the fusion 1/2～2/3 o'clock is got cell conditioned medium and is adopted indirect elisa method to detect antibody titer.The results are shown in following table:

In the present embodiment, cell confluency and positive rate see also following table (fusion rate: expression are merged the hybridoma obtain and detected the ratio of Kong Zhongzhan at all, and the success ratio of reflection mixing operation is generally relevant with the application of integration technology, serum, substratum etc.Positive rate: refer to A in indirect elisa method detects ₄₅₀The hybridoma that value＞negative control is 2.1 times shared ratio in total hybridoma, the immune effect of reflection mouse):

The mouse numbering	The clonal growth hole count is arranged	Fusion rate	Positive rate	Remarks
					A2	768	100％	99.86％	Plate 1-8
A4	457	79.34％	99.78％	Plate numbers 9～14
					B2	576	100％	99.65％	Plate numbers 15～20
B4	380	65.97％	100％	Plate numbers 21～26

4.3 the mono-clonalization of hybridoma

1) flap is cultivated: select 156 holes altogether in the above-mentioned positive colony hole that filters out, go to 24 well culture plates from 96 well culture plates, cultivated in the DEME complete culture solution 3～4 days.

2) repetition measurement: treat on 24 orifice plates that the clone grows at the bottom of the hole at about 1/2 o'clock, get cell conditioned medium and carry out the ELISA repetition measurement that repetition measurement obtains the positive strain of 36 strains altogether.Be GST (-), rPla (+), natural Pla (+).(illustrate that most of knurl strain lost secretion capacity in screening process, this also is the new common problem of hybridoma that produces that merges, so need through repeatedly repeated screening, just can obtain having the cell strain of stably excreting ability).When selecting repetition measurement and the antigen coated hole A of natural Pla ₄₅₀Value is higher, carry out mono-clonalization with the 21 strain knurl strains that it is good that the GST envelope antigen is negative reaction and growth conditions, and all the other positive strains are frozen.

3) adopt limiting dilution assay (Limiting dilution method) to carry out hybridoma cloning.Concrete operations are as follows:

A. the hybridoma of pending cloning is made suspension, trypan blue dyeing counting viable count.

B. carry out continuous multiple dilution with the incomplete nutrient solution of DEME, make it become the suspension that every ml contains 10000,1000,100,10 cell, the complete culture solution that contains 20%FCS is used in last dilution.

C. prepare feeder cell by method noted earlier, add 96 porocyte culture plates by every hole 100 μ l, the every hole of clone cell suspension for the treatment of that contains 10/ml adds 100 μ l, makes it add 1 cell in every hole in theory.

D. after cultivating for 1 week, the hole and the mark that have only 1 clonal growth are picked out in each visual field of microscopy culture hole, wait to clone 1/3～1/2 o'clock that grows at the bottom of the hole, get supernatant and detect antibody titer with indirect elisa method.

E. select and natural Pla reaction A ₄₅₀Be worth higher, with reactionless, the growth conditions good cell strain of GST, change that 24 orifice plates are cultivated and the repetition measurement supernatant is tired, carry out cloning next time and frozen guarantor's kind by above-mentioned steps.

Month f.2～3 after, the frozen hybridoma cell strains of recovering carries out the secondary cloning by above-mentioned steps.

In the present embodiment, (the positive strain result of 3 strains sees also following table to carry out 3 time cloningizations altogether, all the other cell strains are lost secretion capacity in the cloning process), final screening obtains the hybridoma cell strain of the anti-natural Pla albumen of 3 strains energy stably excreting, difference called after hybridoma cell strain 15B in the present embodiment ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄Monoclonal antibody difference called after monoclonal antibody 15B with these hybridoma cell strain secretions ₈, monoclonal antibody 19A ₄, monoclonal antibody 14H ₄

From last table result as can be seen, in the present embodiment, after the 3rd time cloningization, hybridoma cell strain 15B of the present invention ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄Positive rate all reach 100%.

Among the present invention, with described hybridoma cell strain 15B ₈With hybridoma cell strain 19A ₄Be preserved in (address: Chinese Wuhan City Wuhan University, postcode: 430072), wherein, secrete monoclonal antibody 15B of the present invention, Chinese typical culture collection center ₈Hybridoma cell strain 15B ₈Preservation date: on September 16th, 2010, deposit number: CCTCCNO:C201091, classification name: hybridoma cell strain 15B ₈Secrete monoclonal antibody 19A of the present invention ₄Hybridoma cell strain 19A ₄Preservation date: on September 16th, 2010, deposit number: CCTCC NO:C201094, classification name: hybridoma cell strain 19A ₄

5 hybridoma chromosome karyotype analysis

Check the most frequently used colchicine method of the chromosomal method of hybridoma, its principle is to use the special destruction spindle fibre of colchicine and obtain metacinesis phase cell; Use hypotonic processing such as 0.075mol/L KCl solution again, make cell expansion, volume increases, and karyomit(e) is loose; Fix through methyl alcohol-glacial acetic acid solution, get final product observation and inspection.Its operation steps is as follows:

1) 48～36h goes down to posterity hybridoma before adding colchicine.

2) colchicine is handled: add colchicine (100 μ g/ml, degerming ,-20 ℃ of preservations) in culturing bottle, making ultimate density is 0.1～0.4 μ g/ml.37 ℃ are continued to cultivate 4～6h, blow and beat cell then, move in the centrifuge tube, and the centrifugal 10min of 1000r/min abandons supernatant.

3) hypotonic processing: kowtow gently and hit the centrifuge tube bottom, make the cell mixing.Add temperature in advance to 37 ℃ 0.075mol/LKCl solution 5.0ml, with the sedimentation cell also mixing that suspends, the 37 ℃ of hypotonic processing of water-bath 25min.

4) pre-fix: add freshly prepared stationary liquid (methyl alcohol mixes with Glacial acetic acid at 3: 1) 1.0ml in suspension, dropwise add, blow and beat mixing with suction pipe, room temperature is 20min fixedly.

5) fixing: the centrifugal 10min of 1000r/min, abandon supernatant, kowtow gently and hit the centrifuge tube bottom, make cell and remaining solution mixing, slowly add freshly prepared stationary liquid 2～5ml along tube wall, room temperature leaves standstill 30min, and the centrifugal 10min of 1000r/min abandons supernatant.The fresh stationary liquid that adds 10～15 times of cell precipitation volumes in cell precipitation, piping and druming are evenly.

6) film-making: take out centrifuge tube, inhale gently and abandon supernatant liquor, what stay 0.5～1.0ml stationary liquid according to cell pack, behind cell suspension and mixing, draw 1～2 of cell suspension, drop on the slide glass that just from frozen water, takes out, dispel with mouth, and at flame by for several times, make cell be tiled on the slide glass seasoning.

7) dye sheet: with the 10%Giemsa dye liquor dyeing 15～20min of new preparation, with tap water flush away dye liquor, seasoning.

8) microscopy: the selective staining body is scattered, and zero lap does not have the cell that scatters and carries out observation analysis.100 complete metaphase nucleus cells of every part of sample counting, and note observing whether marker chromosome is arranged.

In this enforcement, the colchicine method checks that hybridoma chromosome analysis result shows (referring to Fig. 2), hybridoma cell strain 15B of the present invention ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄The karyomit(e) number average between 95～108, number is substantially near the chromosome number sum of splenocyte and Sp2/0.Analyze from chromosomal form, have the characteristics of hybridoma cell strain: the telocentric chromosome of existing mouse boosting cell has myeloma cell's metacentric chromosom again.

Frozen and the recovery of 6 hybridoma cell strains

6.1 hybridoma is frozen

1) will treat the frozen centrifugal 10min of cell suspension 1200r/min, abandon supernatant liquor.

2) add cells frozen storing liquid (new-born calf serum, DMEM nutrient solution, DMSO were by preparation in 5: 4: 1), be mixed into cell suspension and be sub-packed in the frozen pipe, place in the barrier box.

3) adopt the slow freezing method, be positioned over-20 ℃ of refrigerator 1～2h, spend the night in-70 ℃ of cryogenic refrigerators, move into again in the liquid nitrogen (196 ℃), but prolonged preservation.

6.2 the recovery of hybridoma

During recovery, from liquid nitrogen, take out frozen pipe, melt 37 ℃ of water-baths immediately, when treating that the last point ice cube soon melts, from water-bath, take out, put on the ice bath.With the dilution of 5～10ml complete culture solution, the centrifugal 10min of 1000r/min abandons supernatant, and resuspending changes in culturing bottle or 24 orifice plates in an amount of complete culture solution, puts 37 ℃, 5%CO ₂Cultivate.Can add feeder cell earlier when changing over to.

The preparation of 7 ascites

According to ordinary method, hybridoma is inoculated in the mouse peritoneal to induce generation ascites, concrete operations are as follows:

1) every BALB/c mouse abdominal injection whiteruss 0.5ml.

2) the hybridoma enlarged culturing is grown to logarithmic phase, blow down cell and make suspension, adjust cell density to 1.3 * 10 ⁶～1.4 * 10 ⁶Individual/ml.

3) injecting fluid paraffin is after 7～14 days, every above-mentioned cell suspension 0.5ml of BALB/c mouse abdominal injection, several mouse of every strain of hybridoma injection.

4) after 7～10 days, 1 ascites of extraction in 1～2 day when mouse web portion obviously expands.With the centrifugal 10min of ascites 12000r/min that collects, the compositions such as fat on reject bottom settlings and upper strata, purifying or cryopreservation are stand-by.

8 Purification of Monoclonal Antibodies

According to following operation the collected ascites that contains monoclonal antibody is carried out purifying:

1) presses the appended specification sheets operation of HiTrap Protein G prepacked column (1.0m1).With the sample-loading buffer Binging buffer A balance columns of at least 5 times of column volumes, make in the post solution environmental stable, and make the OD shown in the purifying instrument ₂₈₀The absorbancy baseline is 0 and keeps stable.

2) with ascites with the centrifugal 10min of 12000r/min, remove lipid layer and precipitation, use the dilution of sample buffer B ingingbuffer A equal-volume, remove bubble with 0.45 μ m membrane filtration.

3) the ascites sample with above processing adds injector, uses sample buffer B inging bufferA with sample on the flow velocity of 1.0ml/min.

4) use sample buffer B inging buffer A and wash post by the flow velocity of 2.0ml/min, until the OD shown in the purifying instrument ₂₈₀The absorbancy baseline is 0 and keeps stable.

5) wash post with Elution buffer B by the flow velocity of 2.0ml/min, until the OD shown in the purifying instrument ₂₈₀The absorbancy baseline is 0 and keeps stable.Collect each elution peak.

6) use sample buffer B inging buffer A and wash post, make in the post solution environmental stable, until the OD shown in the purifying instrument ₂₈₀The absorbancy baseline is 0 and keeps stable.

7) each elution peak of SDS-PAGE electrophoresis detection.

8) gained antibody is dialysed more than the 48h with 25mmol/L Tris-C1 pH8.0, change damping fluid therebetween 3 times, the damping fluid volume is at least 20 times of antibody volume.Short-term is used 4 ℃ of preservations, for a long time then packing-20 ℃ preservation in a small amount.Totally 7 inferior strain ascites are through Protein G purifying for 3 strain hybridoma cell strainses, and SDS-PAGE analyzes purification effect, the results are shown in Figure 3.Calculate through software analysis, purity all reaches more than 95%.Tire to detect before the ascites purifying and behind the purifying and see the following form.

Before the ascites purifying, the detected result of tiring behind the purifying

The cell strain numbering	Before the purifying (1 :)	Behind the purifying (1 :)
			14H4a	3.2×10 6	10 4
14H4c	3.2×10 6	10 4
			14H4d	3.2×10 6	10 4
19A4a	1.6×10 6	10 4
			19A4b	1.6×10 6	10 4
15B8a	1.6×10 6	10 4
			15B8b	1.6×10 6	10 4

The preliminary evaluation of 9 monoclonal antibodies

9.1 antibody titer is measured

Measure antibody titer in Hybridoma Cell Culture 72h supernatant and the ascites with indirect elisa method, bag is by concentration natural Pla 5 μ g/ml, HRP-sheep anti mouse working concentration 1: 8000.

The results are shown in following table (lowercase after the knurl strain number represents inferior strain numbering).

Knurl strain number	Supernatant (1 :)	Ascites (1 :)
			15B8a	25600	1.6×10 6
15B8b	12800	1.6×10 6
			19A4a	25600	1.6×10 6
19A4b	25600	1.6×10 6
			14H4a	25600	3.2×10 6
14H4c	25600	3.2×10 6
			14H4d	25600	3.2×10 6

9.2 antibodies specific is identified

1) indirect elisa method detects antibodies specific

Because the immunity of gained hybridoma cell strains is reorganization Pla (rPla) antigen with antigen among the present invention, this albumen is the fusion rotein of being combined with purification tag GST, so when screening, detect as envelope antigen with rPla, natural Pla, three kinds of albumen of GST respectively.

To hybridoma cell strain 15B of the present invention ₈, hybridoma cell strain 19A ₄, hybridoma cell strain 14H ₄Culture supernatant adopts indirect elisa method, and with 3 kinds of antigen coated detections, the result is: natural Pla (+), rPla (+), rGST (-).

2) Western Blotting identifies antibodies specific

A, SDS-PAGE: natural Pla albumen is carried out SDS-PAGE according to a conventional method;

B, film are handled: cut the pvdf membrane onesize with blob of viscose in advance, 40s activates in the immersion methyl alcohol, inserts after the taking-up and changes in the film liquid;

C: balance: electrophoresis after finishing cuts adhesive tape to suitable size, puts into and changes film liquid, will cut out good filter paper simultaneously and also put into, and slowly jolts 20min;

D: change film: be positioned in the commentaries on classics film instrument by the order of placing filter paper, pvdf membrane, gel, filter paper from bottom to top successively, filter paper, gel accurately align with pvdf membrane (or from bottom to top gradually reduce), each step is removed bubble with glass stick, and excess liquid blotted, improvement film instrument is installed, connect power supply, constant voltage 25V shifts 1h.;

E, sealing: pvdf membrane is taken out from change the film instrument, be positioned in the membrane closure liquid, the shaking table sealing is spent the night under the room temperature;

F, wash film 3 times with lavation buffer solution, each 5min;

G, 4 ℃ of own purifying preserve ascites with dilution buffer liquid by dilution in 1: 1000, washed film is put into, liquid needs the whole of mulch film, and 37 ℃ of shaking tables shake 1h;

H, abandon the ascites diluent, wash film respectively 3 times with lavation buffer solution, each 5min;

I, with HRP-SPA with diluted to working concentration (1: 40000), add above-mentioned washed film, 37 ℃ of shaking tables shake 1h;

J, abandon the HRP-SPA working fluid, wash film 3 times with lavation buffer solution, each 5min;

K, film is added the 5min that develops the color in the DAB colour developing liquid, take out film ddH ₂The O flushing, scanner scanning result behind the airing.

In the present embodiment, be primary antibodie with the mouse ascites, HRP-SPA is that the two anti-Western blot that carry out identify; The result shows that 3 strain hybridomas obtain the monoclonal anti physical efficiency and identify two molecular weight bands that extract natural Pla albumen specifically.(because Pla has three kinds of form: α-Pla, β-Pla, γ-Pla, relative molecular mass (Mr) is respectively 37000,35000,31000 to the results are shown in Figure 4.The natural Pla albumen of this experimental applications is through mass spectroscopy (4700 type time-of-flight mass spectrometer MALDI-TOF/TOF-MS, Applied Biosystems, Foster City, CA, USA), in relative molecular mass (Mr) is about 31000,35000 band, contain Pla, thereby two colour developing bands appear in this experiment).

3) antibody subgroup identification

Application is measured test kit available from the mouse monoclonal antibody hypotype of Hyult biotechnology b.v. and is carried out the monoclonal antibody subgroup identification.Concrete grammar: cultivate face one half approximately when hybridoma grows in the DMEM-20%FCS nutrient solution, being changed to ADCF does not gradually have the albuminous cell nutrient solution (ADCF nutrient solution full name is that Animal Derived Component Free. is one of serum-free cell cultures series product of producing of Hyclone company.What this experiment was adopted is the SH3034.02 product, and this nutrient solution contains the 4.6mM glutamine, and 1.0g/L Pluronic F-68 does not contain phenol red) cultivate, collect supernatant after 3～4 days, the centrifugal 10min of 1000r/min removes sediment.Add in the reaction tubes by the test kit requirement with this cell conditioned medium, shake until the appearance of two colour developing points, i.e. antibody subtype and light chain type.The subclass measurement result sees the following form and Fig. 5.Because of 14H ₄, 19A ₄Two strain hybridoma cell strainses belong to different subclass, and in repeated detection this two strains monoclonal antibody tire stable (this two strains hybridoma cell strains through continuous several times go down to posterity and the recovery of 1 month, 3 months liquid nitrogen cryopreservation cell strains after the detection of culture supernatant, the antibody titer unanimity, show and all have good secretion stability), have the applications well prospect.

The antibody subclass detected result that hybridoma produces

Knurl strain numbering	The antibody subclass	The light chain type
			14H 4	IgG2a	K
15B 8	IgG2a	K
			19A 4	IgGl	K

The application of embodiment 2Pla monoclonal antibody

That uses the present invention's foundation can secrete anti-plague Pla protein monoclonal antibody hybridoma cell strain, according to a conventional method, preparation ascites, after ascites is purified, preparation colloidal gold immunochromatographydetection detection test paper bar, the chamber of experimentizing test.

After the recovery of plague EV76 bacterial strain, the cultivation of going down to posterity, preparation is through the bacteria suspension of 28 ℃ of cultivations, and turbidimetry is determined bacterial concentration, is that different concns detects with carbonate buffer solution (pH7.4) dilution.And with F1 Detection of antigen test paper row culture contrast experiment arranged side by side.

The result sees also following table and Fig. 6 A and Fig. 6 B, and Pla Detection of antigen colloidal gold immuno-chromatography test paper strip can detect 28 ℃ of cultivations, bacteria concentration 1 * 10 ⁶The plague EV76 bacterium of individual bacterium/ml (Fig. 6 A), and F1 Detection of antigen colloidal gold strip only can detect to 7 * 10 ⁸The plague EV76 bacterium of individual bacterium/ml (Fig. 6 B).This explanation is not expressed or less expression because of F1 antigen for the plague bacillus of non-37 ℃ of growths, can cause false negative when detecting according to a conventional method, might fail to pinpoint a disease in diagnosis, but Pla can be detected, this method have as the auxiliary diagnosis of the plague may.

Claims (7)

1. one kind has specific monoclonal antibody to plague bacillus plasminogen activating factors Pla albumen, this monoclonal antibody is the hybridoma secretion of CCTCC NO:C201091 or CCTCC NO:C201094 by preserving number, the antibody type of monoclonal antibody that by preserving number is the hybridoma secretion of CCTCC NO:C201091 belongs to IgG2a, is that the antibody type of monoclonal antibody of the hybridoma secretion of CCTCC NO:C201094 belongs to IgG1 by preserving number.

2. monoclonal antibody according to claim 1, this antibody has specificity to natural Pla albumen, and glutathione s transferase is not had specificity.

3. secrete the hybridoma cell line of the described monoclonal antibody of claim 1, its preserving number is CCTCC NO.C201091 or CCTCC NO.C201094.

4. the described monoclonal antibody of claim 1 is for the preparation of the application in the preparation that detects plague bacillus and/or the auxiliary diagnosis plague.

5. preparation that detects Pla albumen, said preparation contains the described monoclonal antibody of claim 1.

6. a colloidal gold immuno-chromatography test paper strip that detects Pla albumen contains the described monoclonal antibody of claim 1 on this colloidal gold immuno-chromatography test paper strip.

7. a colloidal gold immuno-chromatography test paper strip that detects plague bacillus and/or the auxiliary diagnosis plague contains the described monoclonal antibody of claim 1 on this colloidal gold immuno-chromatography test paper strip.

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