CN104388392B - A kind of enrofloxacin monoclonal antibody and its preparation method and application - Google Patents

Abstract

The invention provides a kind of enrofloxacin monoclonal antibody and its preparation method and application.Illustrate, the present invention makees immunogene and envelope antigen simultaneously with Enrofloxacin and its with the conjugate of Cationic bovine serum albumin, indirect chessboard ELISA method is set up to demarcate suitable envelope antigen concentration and antiserum working concentration, affinity of the primary antibody to be measured to Enrofloxacin standard specimen is analyzed through indirect competitive ELISA again, is more than 1 with antiserum working concentration:50000 and 503nhibiting concentration IC₅₀No more than 5ng/ml selects candidate mouse as index, obtains hybridoma cell strain 14H4A1 and 14F8B1 by screening using cell-fusion techniques and through strict, two plants of cells induce ascites, and then obtain target monoclonal antibody.The enrofloxacin monoclonal antibody with high titre and high-affinity that the present invention is provided, realizes the actually detected of Enrofloxacin denier residual, to realize that efficiently quick detection provides technical support and material base at scene.

Description

A kind of enrofloxacin monoclonal antibody and its preparation method and application

Technical field

The invention belongs to immunochemical technique field, and in particular to high titre, high-affinity and high specific Enrofloxacin monoclonal antibody and preparation method thereof, the application of enrofloxacin residual, Yi Jifen are detected using the monoclonal antibody Secrete the hybridoma cell strain for producing the monoclonal antibody.

Background technology

Enrofloxacin (Enrofloxacin), also known as ENR, are the special fluoroquinolones of first livestock and poultry Medicine, have the advantages that has a broad antifungal spectrum, antibacterial activity are strong because of it, easily absorb, be distributed in vivo it is wide be widely used in animal doctor and face Bed prevention and treatment.Because the medicine is remained in animal food and induces this problem of human disease bacterium generation drug resistance It was found that, the Ministry of Agriculture of China provide Enrofloxacin in muscle, milk MRL (Maximum Residue Limit, MRL 100 μ g/kg) be must not exceed.Therefore, strengthen seeming to residue detection of the medicine in animal food and supervision and management It is especially prominent.

However, as the Enrofloxacin of haptens body can not be induced to produce antibody in itself, it is necessary to by itself and carrier protein Coupling, which obtains artificial synthesized comlete antigen, just has antigenicity.From Japanese scholars Hiroo Watanabe in 2001^[1]Deng first Prepare specific enrofloxacin monoclonal antibody and effective detection has gone out in food samples since enrofloxacin residual, it is domestic with eating The safety-related scientific research institution of product has carried out the development work of Enrofloxacin immunity detection reagent in succession.2006-2009, Liu Chun Pretty young woman, Liu Hong, Wang Zhenhui, Jiang Xingdong etc.^[2-7]Report the foundation of Enrofloxacin immunologic detection method and to grace in animal husbandry and fishery product The detection work of promise sand star residual；2006-2012, Tang Hong, Shen Jianzhong, Lu Maolin, Sun Yuanming, Wu Jianxiang etc.^[8-12]Declare The national patent related to Enrofloxacin immune detection.The studies above achievement is shown：The country is to Enrofloxacin detection sensitivity IC₅₀ Only 10-50ppb, test limit IC₂₀For 1-10ppb, effective detection concentration span (IC₂₀-IC₈₀) (represent detection for 1-100ppb Precision), but these detect parameters and not fully up to expectations, and then it is difficult to convert into practical application.In contrast to this, with Germany Biopharm companies for the Ennoshaxing ELISA detection reagent kit of the foreign trans-corporation of representative then there is more excellent detection to join Number, its detection sensitivity IC₅₀For 1ppb, effective detection concentration span (IC₂₀-IC₈₀) it is 0.2-5ppb, almost monopolize the country Market.Lu Maolin etc. substitutes conventional horseradish peroxidase-labeled with time resolved fluoro-immunoassay and improves kit Test limit.But this method is only exaggerated signal intensity, but without fundamentally parent of the raising correspondence antibody to haptens And power, while expanding effective detection concentration span, greatly reduce accuracy of detection；More disadvantageously, these technologies Using senior detecting instrument or equipment is generally required as hardware support, the problems such as additionally, there may be traction is serious to make About this method using and promotes.

In consideration of it, research and development same level detection product has become the task of top priority, need badly a kind of with high titre, high-affinity And the enrofloxacin monoclonal antibody of high specific come meet industry and daily life needed for.

The content of the invention

Low for existing domestic Enrofloxacin immunity detection reagent detection sensitivity, false positive rate is high, surveys sample master The state of the art of dependence on import detection kit is wanted, it is an object of the invention to provide a kind of potency height, high specificity, detection spirit High enrofloxacin monoclonal antibody of sensitivity and preparation method thereof, and then to develop level with equal to even better than imported product Immunity detection reagent lay the foundation.

It is another object of the present invention to provide the hybridoma cell strain for secreting the enrofloxacin monoclonal antibody.

The purpose of the present invention is achieved by the following technical programs：

Secretion produces the hybridoma cell strain 14H4A1 or 14F8B1 of enrofloxacin monoclonal antibody in the present invention；Wherein： 14H4A1 is mouse hybridoma cell system CCTCC NO.C201487, and Wuhan City, Hubei Province China is preserved on May 6th, 2014 Type Tissue Collection (referred to as CCTCC, positioned at Wuchang Luo Jia Shan Wuhan University in the school), preserving number is CCTCC NO.C201487；14F8B1 is mouse hybridoma cell system CCTCC NO.C201488, and Hubei is preserved on May 6th, 2014 Province's Wuhan City's China typical culture collection center, preserving number is CCTCC NO.C201488.

Cell line numbers explanation：For 14H4A1 plants, 14H4 represents fused cell growth No. 14 culture plate of clone H columns the 4th row growth hole be positive colony hole, A represent the clone carry out first round subclone after respectively numbering be the two of A and B A strains in individual subclone, 1 to represent be that A plant cells are carried out during two of numbering 1 and 2 are subcloned respectively after the second wheel subclone No. 1 strain；14F8B1 plants of coding rule is similar with this.

By mouse hybridoma cell strain 14H4A1 or 14F8B1 secrete produce enrofloxacin monoclonal antibody 14H4A1 ' or 14F8B1’。

The enrofloxacin monoclonal antibody 14H4A1 ' or 14F8B1 ' of the present invention preparation method, it comprises the following steps：

1) preparation of immunogene：Enrofloxacin (ENR) and bovine serum albumin(BSA) (BSA) are coupled, immunogene grace promise is prepared Sha Xing-bovine serum albumin(BSA) conjugate (ENR-BSA)；

2) animal immune is injected：Using mouse as animal is exempted from, according to immune programme for children injecting step 1) in obtain it is immune The former mixture with adjuvant is immunized, and gathers serum sample；

3) candidate is established by mouse is exempted from：Using chessboard ELISA and indirect competitive ELISA detecting step 2) the middle serum obtained Sample, selects and is exempted from mouse, abdominal cavity injection booster immunization for the candidate of cell fusion；

4) acquisition of hybridoma cell strain：Take step 3) in obtain candidate entered by the splenocyte for exempting from mouse with myeloma cell Row cell fusion, screening positive clone, then two wheel subclones are carried out to positive colony, obtain secretion enrofloxacin monoclonal antibody Hybridoma cell strain 14H4A1 or 14F8B1；

5) acquisition of monoclonal antibody：Using abdominal cavity injection system, to mouse injecting step 4) the middle secretion grace promise obtained The hybridoma cell strain 14H4A1 or 14F8B1 of husky star monoclonal antibody, gather ascites, using protein G resin pair after 7~10 days Ascites is purified, and produces enrofloxacin monoclonal antibody 14H4A1 ' or 14F8B1 '.

In the above-mentioned technical solutions, step 1) described in coupling using carbodlimide method complete.

In the above-mentioned technical solutions, step 2) described in adjuvant include Freund's complete adjuvant (CFA) and Freund and not exclusively help Agent (IFA).

In the above-mentioned technical solutions, step 2) described in immune programme for children it is as described below：Setting injection immune each time Amount is 100 μ g/ only, and the time interval between being immunized twice is 4 weeks；Immunogene is emulsified with isometric not formula Freund's complete adjuvant Carried out afterwards using abdominal cavity injection system immune for the first time；The immune mode of third time and dosage are identical with first time, wherein adjuvant It is incomplete Freund's adjuvant, it is 1 with the volume ratio of immunogene:3；5th time immune mode and dosage is identical with first time, Wherein adjuvant is incomplete Freund's adjuvant, and it is 1 with the volume ratio of immunogene:7, while adding the En Nuo of dosage for 50 μ g/ only Husky asterisk sample；Second and the 6th immune mode be subcutaneous multi-point injection, wherein adjuvant is isometric not with immunogene Family name's Freund's incomplete adjuvant；4th immune mode is subcutaneous multi-point injection, wherein adjuvant be the Freund isometric with immunogene not Freund's complete adjuvant, while adding the Enrofloxacin standard specimen of dosage for 50 μ g/ only.

In the above-mentioned technical solutions, step 3) described in the standard selected to carry out bioactivity in indirect ELISA experiment Working concentration be more than 1:50000 and indirect competitive ELISA experiment in detection serum to the inhibition value IC of Enrofloxacin₅₀It is small In equal to 5ng/ml.

In the above-mentioned technical solutions, step 4) described in myeloma cell be Sp2/0Ag14 murine myeloma cells, with PEG1500 is as fusion agent, according to splenocyte:Myeloma cell=8:1 ratio carries out cell fusion.

In the above-mentioned technical solutions, step 5) in mouse peritoneal injection secrete ENR monoclonal antibodies hybridoma The injection volume of strain is 1~2 × 10⁶Individual/only.

Prepared it is also another object of the present invention to provide above-mentioned enrofloxacin monoclonal antibody for detecting Enrofloxacin Reagent in purposes, it, which is mainly used in, prepares enrofloxacin residual detection kit and colloidal gold strip.

Due to the use of above-mentioned technical proposal, compared with prior art, the invention has the advantages that：

1. the present invention uses immunogene with detection antigen (envelope antigen) for same haptens and protein carrier conjugate Policy Filtering antibody, the strategy not only greatly reduce the difficulty of haptens and protein carrier conjugate chemical synthesis with into This, while adding protein carrier selectivity, this method can be commonly used in the preparation of other haptens correspondence antibody；

2. the monoclonal antibody hybridoma cell strain 14H4A1 or 14F8B1 that the method provided using the present invention is obtained, its point The titre for secreting Enrofloxacin antibody is high, and wherein 14H4A1 plants of culture supernatant working concentration is 1:1,600, induce ascites work dense Spend for 1:160,000；14F8B1 plants of culture supernatant working concentrations are 1:800, it is 1 to induce ascites working concentration:75,000；

3. the monoclonal antibody hybridoma cell strain 14H4A1 or 14F8B1 secretions that the method provided using the present invention is obtained Monoclonal antibody it is high to Enrofloxacin standard specimen affinity, 503nhibiting concentration IC of two plants of cell secretory antibodies to Enrofloxacin₅₀ Respectively 0.3 and 0.9ng/ml；

4. the monoclonal antibody hybridoma cell strain 14H4A1 or 14F8B1 secretions that the method provided using the present invention is obtained Monoclonal antibody secretion antibody to Enrofloxacin high specificity, (such as Ciprofloxacin, promise fluorine are husky with Enrofloxacin analog Star, Ofloxacin) between cross reacting rate be respectively less than 0.50%；

5. the monoclonal antibody hybridoma cell strain 14H4A1 or 14F8B1 secretions that the method provided using the present invention is obtained Monoclonal antibody secretory antibody stability it is strong, two plants of cells expand its point after culture in the generation of continuous passage ten and cryopreservation resuscitation Secrete antibody titer and the affinity binomial index to Enrofloxacin is basically unchanged.

Brief description of the drawings

Fig. 1 is the purple of Enrofloxacin (ENR), Cationic bovine serum albumin (BSA) and the two conjugate (BSA-ENR) Outer microphotohram.

Fig. 2 is 14 in hybridoma selection course^#The ELISA colour developing result figures of plate.

Fig. 3 is 4 in Enrofloxacin specific antibody positive colony elaboration process^#Plate colour developing result figure.

Fig. 4 is the competitive ELISA result figure for the single cell clone culture supernatant that 14H4 cell lines three-wheel is subcloned.

Fig. 5 is the suppression curve of the antibody that produces to Enrofloxacin in two strain of hybridoma culture supernatants.

Fig. 6 is suppression curve of the antibody to Enrofloxacin that two strain of hybridoma induce ascites.

Fig. 7 is that the sample SDS-PAGE after 14H4A1 monoclonal antibody protein wash fraction collections analyzes and identifies figure.

Embodiment

The present invention is described in more detail with specific embodiment below in conjunction with the accompanying drawings.It should be noted that this Description in a little embodiments is only used for making those skilled in the art more fully understand the present invention, and not limits the model of the present invention Enclose.

Embodiment one：The preparation and detection of Enrofloxacin immunogene.

Specific experiment step prepared by immunogene is as follows：

1. the closing of carrier protein：By ethylenediamine (EDA) (20 μ l), bovine serum albumin(BSA) (BSA) (23.6mg), two hexamethylenes Base carbodiimide (EDC) (25.6mg) is dissolved in pH=7.0 0.01M PBS liquid (1mL) successively, in coupling agent EDC work Under, the residual carboxylic acid group in closing BSA molecules, shaken overnight (20 DEG C, 110rpm), using PBS 24h, removes excess EDA And EDC, obtain cationization BSA standby.

2. the coupling of Enrofloxacin and carrier protein：Enrofloxacin standard specimen (ENR) (15.3mg), N- hydroxyls oxalyl is sub- Amine (NHS) (5.2mg), EDC (9.3mg) are dissolved in DMF (DMF) (500 μ l), and room temperature lucifuge vibrated At night (20 DEG C, 110r/min), form Acibenzolar；5min (3000r/min) is centrifuged, takes supernatant (100 μ l) to add cationization BSA liquid (2ml, wherein containing 5mg cationizations BSA), room temperature lucifuge shaken overnight (20 DEG C, 110r/min)；Centrifuge (3000r/ Min) 5min, takes supernatant to be placed in bag filter, respectively with 33%, 28%, 23%, 18%, the 13% of 100X ice-water bath precoolings DMF/PBS respectively dialyses after 4h, then stayed overnight with PBS.Take a little progress ultraviolet spectrometry (surveying 200-400nm peaks) and SDS-PAGE Electroresis appraisal, remainder is prepared into freeze-dried powder, and estimates salt ratio.

By the above method, about 100mg (in terms of protein content) Enrofloxacins and cationization bovine serum albumin are obtained altogether The freeze-dried powder of white conjugate (ENR-cBSA), uv-spectrophotometric figure as shown in Figure 1 is shown：Enrofloxacin standard items exist There is obvious characteristic absorption peak at 270nm and 330~350nm；Cationization BSA has obvious protein specificity to inhale at 278nm Peak is received, and without obvious absorption peaks at 330~350nm；Conjugate ENR-cBSA is at 276~278nm and at 330~350nm There is obvious characteristic absorption peak, the result shows that Enrofloxacin is successfully coupled on protein molecular, through [OD₂₇₈/ BSA molecules Amount]/[OD₃₃₄/ (3.4X ENR molecular weight)] formula calculating：Enrofloxacin is 13 with BSA mol ratios:1, meet immunogene Basic demand.Meanwhile, Enrofloxacin and untreated bovine serum albumin(BSA) conjugate are prepared using same method, two are found Person's mol ratio is only 3:1, the two, which compares, shows that the cationization processing of bovine serum albumin(BSA) is to improve hapten conjugation rate to have Efficacious prescriptions method.

Embodiment two：Animal immune and antiserum detection.

1. animal immune：Using BALB/c mouse as immune animal, it is immunized according to the program in table 1.

The mouse immune proceedings table of table 1.

Wherein：E-B represents ENR and cBSA conjugates；E represents ENR；

CFA represents Freund's complete adjuvant；IFA represents incomplete Freund's adjuvant.

Setting injection dosage immune each time is 100 μ g/ only, and the time interval between being immunized twice is 4 weeks；Exempt from Epidemic focus is immune for the first time with using abdominal cavity injection system to carry out after isometric not formula Freund's complete adjuvant emulsification；The immune side of third time Formula and dosage are identical with first time, and wherein adjuvant is incomplete Freund's adjuvant, and it is 1 with the volume ratio of immunogene:3；5th time Immune mode and dosage is identical with first time, and wherein adjuvant is incomplete Freund's adjuvant, and it is 1 with the volume ratio of immunogene: 7, while adding the Enrofloxacin standard specimen of dosage for 50 μ g/ only；Second and the 6th immune mode be subcutaneous multi-point injection, Wherein adjuvant is the incomplete Freund's adjuvant isometric with immunogene；4th immune mode is subcutaneous multi-point injection, wherein Adjuvant is the incomplete Freund's adjuvant isometric with immunogene, while adding the Enrofloxacin standard specimen of dosage for 50 μ g/ only.6th 7-10 days after secondary immune end, mouse serum sample is exempted from collection, is detected serum titer using chessboard indirect elisa method, is adopted simultaneously Inhibition (i.e. IC of the serum to Enrofloxacin is detected with indirect competitive ELISA method₅₀).Choose the high (working concentration of serum titer More than 1:50000) and to the strong (IC of Enrofloxacin affinity₅₀No more than 5ng/ml) mouse, after rest 4 weeks, then noted with abdominal cavity The mode of penetrating adds booster immunization once, and dosage is 200 μ g/, wherein using ENR-BSA as immunogene, and assistant is not added Agent (the 7th time immune).

2. chessboard indirect elisa method demarcation antiserum titre is comprised the following steps that with suitable envelope antigen concentration：

(1) it is coated with the preparation of buffer solution：Weigh Na₂CO₃(1.59g) and NaHCO₃(2.93g), plus tri-distilled water dissolve and determined Hold to 1000mL, obtain 0.05M Na₂CO₃/NaHCO₃It is coated with buffer solution (pH=9.6)；

(2) antigen coat：Using coating buffer solution dilution envelope antigen ENR-cBSA, (coupling ratio is 13:1), it is coated with concentration It is 9,3,1,0.333,0.111,0.037,0.012 μ g/ml (being coated in the A-G columns of elisa plate, 100 μ l/ holes) respectively；Coating Antigen control：Cationic bovine serum albumin (cBSA), is coated with concentration：3 μ g/mL (are coated in the H columns of elisa plate, 100 μ l/ Hole), in 37 DEG C it is wet incubate after 1~1.5h, then be placed in wet in 4 DEG C of refrigerators incubate overnight；

(3) preparation of PBS liquid：Weigh Na₂HPO₄·12H₂O(2.9009g)、NaH₂PO₄·2H₂O(0.2964g)、NaCl (8.5g), plus tri-distilled water dissolve and are settled to 1000ml, obtain 0.01M PBS cleaning solutions (pH=7.4)；

(4) wash：Coating plate is taken out, coating buffer is dried, 3 times (300 μ l/ holes) is washed with PBS liquid, and clapped on blotting paper It is dry；

(5) close：Dosage according to 340 μ l/ holes doses 1% oralbumin (OVA, Ovalbumin)/PBS closings Liquid, wet 2.5h is incubated in 37 DEG C；

(6) wash ibid；

(7) preparation of primary antibody dilution：High-purity O VA (5mg) and cBSA (2.5mg) are weighed, the above-mentioned PBS of 20ml are added to Fully dissolved in liquid, OVA (0.25mg/ml)+cBSA (0.125mg/ml)/PBS liquid is made, and save backup in 4 DEG C；

(8) antibody dilution and pretreatment：Take by the serum for exempting from mouse, be diluted to above-mentioned serum respectively using primary antibody dilution 1:1000、1:3000、1:9000、1:27000 and 1:81000 5 dilution factors, concurrently set 1:3000 be that normal mouse serum is cloudy Property control.Above-mentioned each treatment fluid sample is placed in after shaken at room temperature in decolorization swinging table (100r/min) 1h, primary antibody Incubating Solution is used as It is standby；

(9) preparation of the PBST liquid as cleaning solution：To add in 0.01M PBS liquid (pH=7.4) 0.05% tween- 20, mixing；

(10) wash：Plate is taken out, dries, is washed with PBST liquid 3 times, and patted dry on blotting paper；

(11) primary antibody is incubated：Primary antibody Incubating Solution is separately added into elisa plate 1-2,3-4,5-6,7-8,9-10,11-12 row Hole in, per the μ l of hole 100, wet 2h is incubated in 37 DEG C；

(12) preparation of secondary antibody Incubating Solution：1 is pressed with 0.1%OVA/PBS liquid:500 dilution ELIAS secondary antibody (goat-anti Mouse IgG (H+L) ELIAS secondary antibody)；

(13) wash：Withdrawing plate, dries, is washed with PBST liquid 5 times, and patted dry on blotting paper；

(14) secondary antibody Incubating Solution is added with the dosage in 100 μ l/ holes, wet 1.5h is incubated in 37 DEG C；

(15) wash：Withdrawing plate, dries, is washed with PBST liquid 4 times, then is washed 2 times with PBS liquid, and is clapped on blotting paper It is dry；

(16) preparation of ELISA nitrite ions：By 0.1M citric acid solutions (4.86ml) and 0.2M Na₂HPO₄Liquid (5.14ml) is mixed Close, add after o-phenylenediamine (OPD) (6mg) fully dissolving, add 30% H₂O₂(20 μ l), lucifuge is standby；

(17) develop the color：After drying, ELISA nitrite ions, lucifuge colour developing 3min, then per hole are added with the dosage in 100 μ l/ holes Add 0.2M H₂SO₄(50 μ l) is with terminating reaction；

(18) measurement OD values (at 490nm).

Result judgement：With the OD values in Sample serum hole in the antibody corresponding to hole of 1.0 or so the and P/N values more than 2.1 For the potency of the serum, (P represents the OD values in measuring samples serum hole to dilution factor；N represents the OD values in negative control sera hole).

3. antiserum to the indirect competitive ELISA method of Enrofloxacin to set up process as described below：

(1) it is coated with the preparation of buffer solution：Ibid

(2) antigen coat：Envelope antigen is used as by the ENR-cBSA of above-mentioned indirect ELISA Experimental Calibration concentration and is coated with enzyme The A-G columns (100 μ l/ holes) of target；Envelope antigen is compareed：With the H columns (100 μ l/ holes) of concentration c BSA coated elisa plates, in 37 DEG C It is wet to incubate 1~1.5h, then be placed in 4 DEG C and wet incubate overnight；

(3) washing-closing-wash again ibid；

(4) the pre- combination of antibody and free haptens：

(a) antiserum is diluted to 2 times of demarcation antiserum dilution factors with 0.25mg/ml cBSA+0.1mg/ml OVA/PBS It is standby as working concentration antibody；

(b) appropriate Enrofloxacin standard specimen is weighed, 100 μ g/ml are diluted to 0.03M NaOH liquid, makees standby (4 DEG C of storing liquid Under can place one month)；

(c) 7 5ml centrifuge tubes are taken, above-mentioned Enrofloxacin storing liquid is diluted respectively with 0.01M PBS liquid (pH=7.4) Into 0.2,0.6,1.8,5.4,16.2, six dilution factors of 48.6ng/ml, and be added separately to wherein 6 centrifuge tubes by 0.7ml/ pipes In, 1.4ml 0.01M PBS liquid (pH=7.4) is added in remaining 1 centrifuge tube, is then added in equal volume in each centrifuge tube Above-mentioned working concentration antibody；

(d) above-mentioned centrifuge tube shaken at room temperature (50~100r/min) is incubated 1h；

(5) primary antibody is incubated：Primary antibody Incubating Solution correspondence comprising free Enrofloxacin competition antigen in above-mentioned 6 test tubes is added Enter into A-F columns (100 μ l/ holes) each column of above-mentioned ELISA Plate, the G of ELISA Plate in H columns plus PBS liquid processing primary antibody Incubating Solution (100 μ l/ holes), wet 2h is incubated in 37 DEG C；

(6) washing, secondary antibody after primary antibody is incubated are incubated, wash, developed the color with above-mentioned indirect ELISA method.

Interpretation of result：ELIASA is read per hole OD₄₉₀Value, averages, the average value on A-G columns subtracts H columns average value by column The actual value after background values is removed as each column afterwards, each concentration competition antigen processing column actual value is named as B, uncontested antigen G columns actual value is named as B₀, the inhibiting rate per column is by (B₀-B)/B₀× 100% calculates, then the concentration for competing antigen with every column is made Independent variable, the inhibiting rate per column makees dependent variable and draws suppression curve, and insertion obtains IC₂₀、IC₅₀、IC₈₀(inhibiting rate is respectively 20%th, 50%, 80% corresponding Enrofloxacin concentration) three critical concentration points, wherein IC₅₀The detection for representing antibody sample is sensitive Degree, IC₂₀Represent the highest detection limit of antibody sample, IC₂₀-IC₈₀Represent the effective detection concentration range of the antibody sample.

4. six times it is immune after by exempting from Mouse Antisera titre and as follows with the affinity result of Enrofloxacin：

Exempted from mouse six for 6 and exempt from one week after, take its antiserum sample respectively, and sample carried out respectively chessboard ELISA and Competitive ELISA experiment is connect, 6 Mouse Antisera samples are through hybridization signal intensities OD in chessboard ELISA experiments₄₉₀For 2.0~2.5 when Envelope antigen concentration and the dilution factor of primary antibody to be measured are shown in Table 2.Table 2 shows：2^#Mouse and 3^#The antiserum titre highest of mouse.

The hybridization signal intensities OD of table 2.₄₉₀For 2.0~2.5 when envelope antigen concentration and primary antibody to be measured dilution factor

Competed with envelope antigen and the appropriate dilution primary antibody to be measured of suitable concn, then with the Enrofloxacin of series concentration Antigen indirect competitive ELISA is tested.As a result show, the specificity that body has produced anti-Enrofloxacin molecular antigen epitope resists Body, is respectively shown in Table 3 by Mouse Antisera is exempted to effective inhibiting rate of haptens and haptens 503nhibiting concentration.Table 3 shows：2^#Mouse and 3^# The antiserum of mouse is high to Enrofloxacin affinity.Therefore, 2 are chosen^#Mouse is in 3^#Mouse is used for the system of monoclonal antibody as candidate mouse It is standby.

Table 3. is by exempting from Mouse Antisera to effective inhibiting rate of haptens and haptens 503nhibiting concentration

Embodiment three：Cell fusion, filtering hybridoma and colonized culture.

Three days after 7th booster immunization, extracting spleen cell carries out cell fusion, chooses positive colony and is subcloned, is obtained It is capable of the hybridoma cell strain of stably excreting monoclonal antibody, specific experiment process is as described below：

1. cell fusion and culture：

(1) preparation of myeloma cell：The fusion same day takes 4~5 bottles (50ml standard cell lines bottles) to be in exponential phase Sp2/0Ag14 murine myeloma cells, gently patting bottle wall makes cell detachment and is collected in 50ml sterile centrifugation tubes, 1000rpm centrifugations 5min removes culture medium, plus the washing of serum-free RPMI-1640 nutrient solutions, and centrifugation once, then uses 20ml serum-frees RPMI-1640 nutrient solutions are resuspended, and cell count is standby；

(2) preparation of feeder cells：The previous day execution mouse and alcohol disinfecting are merged, is cultivated with serum-free RPMI-1640 Liquid eluriates mouse peritoneal, takes macrophage, and 1000rpm centrifugations 10min removes nutrient solution, suspended with 1 times of HAT Selective agar medium thin Born of the same parents, and by 2 × 10⁴The μ l of individual cell/100 are inoculated in 96 porocyte culture plates, are incubated at CO₂In incubator；

(3) preparation of splenocyte：2 after exempting from six^#Mouse, 3^#Mouse is as candidate mouse, and every 28 days, booster immunization once, was exempted from again Eyeball blood sampling is plucked within 3 days after epidemic disease, positive antibody control is further analyzed or made to antiserum for antibody, after the neck that breaks is put to death, by the mouse It is soaked in 75% alcohol and sterilizes, it is sterile to take spleen, connective tissue is removed, splenocyte suspension is prepared, is transferred in centrifuge tube, is used RPMI-1640 nutrient solutions wash connective tissue, and centrifugation abandons supernatant, plus RPMI-1640 nutrient solutions to 30mL after merging, counts standby With；

(4) cell fusion：By splenocyte:Myeloma cell is about 8:1 ratio is mixed, after gently mixing, 1000rpm centrifuges 10min, removes culture medium, and bullet pine cell precipitation block is placed in centrifuge tube in the 37 DEG C of tepidarium that sterilizes, 1ml is added dropwise 37 DEG C of pre-temperature PEG1500, stir 90s, in then five minutes plus 10ml 37 DEG C of pre-temperature RPMI-1640 culture mediums dilution fusion Cell, specifically distributing is：The first two minute, by 1ml/min speed addition culture medium (stirring while adding), presses for latter two minutes 1.5ml/min speed adds culture medium (stirring while adding), and last minute adds 5ml culture mediums (stirring while adding), 800rpm 7min is centrifuged, culture medium is removed, bullet pine cell precipitation block gently adds selective mediums of the 5-10ml containing HAT, gently shaken Afterwards, it is then added in the above-mentioned selective mediums containing HAT of the 150ml of 37 DEG C of pre-temperatures, prepares fused cell suspension；

(5) cell culture：Above-mentioned cell suspension is added dropwise in 15 pieces in advance with individual layer feeder cells by 100 μ l/ holes In 96 porocyte culture plates, in CO₂Cultivated in incubator, liquid is partly changed using HAT (1X) culture medium respectively in the 4th day, the 7th day Once, liquid is partly changed once using HT (1X) culture medium in the tenth day, when hybrid cell growth clone it is long to cell plate hole 1/10~ Supernatant detection screening is taken during 1/4 area in time.

2. the selection and antibody test of hybridoma：

(1) that secretes Enrofloxacin specific antibody is suspected to be positive hybridoma cell clone's acquisition：With between above-mentioned antiserum Connect suitable concn envelope antigen (ENR-cBSA) wrapper sheet of ELISA Experimental Calibrations, the work such as antigen coat, washing, closing and washing Sequence is with above-mentioned antiserum indirect elisa method；

Respectively take above-mentioned fused cell to grow the culture supernatant (100 μ l/ holes) in hole, 96 new hole cell culture are added on one by one In each hole of plate, setting number hole Sp2/0Ag14 cell culture supernatants are used as primary antibody negative control, number hole 1:10000 antiserums are made For primary antibody positive control, detection order table is carried out, adds 10 μ l cBSA/PBS (1mg/ml) per hole, then culture plate is placed in decolouring On shaking table, shaken at room temperature (50~100r/min) is incubated 1h；

Above-mentioned Incubating Solution correspondence is moved in above-mentioned ELISA Plate hole by 100 μ l/ holes and makees primary antibody incubation, remaining step is with anti- Serum indirect elisa method.After colour developing is terminated, with positive control (OD₄₉₀3.0) and negative control (OD value is more than₄₉₀Value is less than or equal to 0.2) making the index verification detecting system can rearward, then by OD₄₉₀Value is suspected to be positive hybridoma cell more than 3.0 hole interpretation candidates Clone, and the cell is expanded one by one be incubated in each hole of 24 well culture plates；

With 2^#Mouse, 3^#Mouse makees candidate mouse, and booster immunization is after 3 days again, extracting spleen cell and Sp2/0Ag14 cell fusions, altogether Plant and carry out HAT medium cultures in 15 piece of 96 porocyte culture plates, the 11st day microscopy finds that obtaining 4000~5000 altogether melts Cell growth clone (average 3-4 clone/hole) is closed, supernatant indirect ELISA testing inspection is taken to OD within the 12nd day₄₉₀Value is more than Totally 49 hole equal to 3.0, takes one of growth plate (14^#), corresponding ELISA (14^#) test data is shown in Table 4, its result that develops the color As shown in Figure 2.

According to the data result that " OVRFLW " (representing numerical value more than 4.2) is shown as in table 4, from the plate (14^#Plate) in look for Go out three corresponding holes：14B5,14F8,14H4, and preliminary interpretation is is suspected to be positive colony hole, and correspondingly breed thin in 24 holes In born of the same parents' cultivation plate hole, other growth plates are equally handled.

Table 4.14^#The ELISA test datas of corresponding aperture in growth plate

(2) the further establishment of Enrofloxacin specific antibody positive colony：

Envelope antigen is coated in ELISA Plate by upper method, coating-washing-closing-is standby after washing again (method is ibid).

It is suspected to be that positive hole is expanded in 24 well culture plate holes one by one by above-mentioned 49, cell to be amplified is in exponential phase When, respectively take culture clone's supernatant to make primary antibody to be measured respectively, above-mentioned each supernatant sample is divided into two parts, and portion is former for 300 μ l supernatants Liquid, then the liquid is divided into two equal portions, add to respectively in two 1.5ml EP pipes；Another is 30 μ l supernatant stostes, plus 270 μ l PBS is diluted to 1:10 supernatant dilutions, are then separated into two equal portions, add in two 1.5ml EP pipes, the one of supernatant stoste Pipe plus isometric 0.2mg/ml cBSA/PBS；Another pipe of supernatant stoste adds isometric 0.2mg/ml cBSA+ 2000ng/ml ENF/PBS；1:10 supernatant dilution pipes are equally handled, i.e., one supernatant sample, four processing：Stoste+PBS, it is former Liquid+ENF, dilution+PBS, dilution+ENF, above-mentioned processing tube are placed in decolorization swinging table shaken at room temperature (50-100r/min) and incubated 1h is educated, makees primary antibody Incubating Solution respectively standby.

The above-mentioned elisa plate for being surrounded by envelope antigen is divided into two Ge great areas：A-D columns are the firstth area, and E-H columns are the secondth area. By each processing adds a line in a supernatant sample and often the form of two multiple holes of row is added in two Ge great areas of elisa plate respectively In each columns of A-D or E-H, i.e., one clone's supernatant sample adds eight test holes to make primary antibody incubation, and remaining step is indirect with antiserum ELISA method.After colour developing is terminated, ELIASA is read per hole OD₄₉₀Value.

Compared between first being organized to the stoste+PBS groups of each sample with stoste+ENR：Must have very with stoste+PBS groups Strong hybridization signal is used as an important indicator (OD₄₉₀4.0 are have to be larger than, is worth and is preferentially screened for overflow person), compared with upper group Stoste+ENR group signals have obvious decrease.Weaken degree is bigger to prove that specificity is stronger, preferential screening.

Consider further that antibody titer is likely to be at supersaturation in supernatant sample, antigen can be to sample to be tested although ENR makees competition Depression effect is produced, but because the detection range estimated with ELISA detectors has limitation, it is impossible to substantially measuring the effect can Can property.Therefore, this experiment devises 1 simultaneously:The parallel test of 10 dilution supernatants, then it is diluted liquid+PBS groups and dilution Compare between+ENR group groups：And remain to keep very strong hybridization signal (OD with dilution+PBS₄₉₀The even overflow more than 3.5), Compared dilution+ENR group signals with upper group and decline more obvious (OD₄₉₀It is excellent less than 1, or even close to background (0.3) result of the test First screen.

By These parameters comprehensive analysis, 4 Enrofloxacin specificity are filtered out from above-mentioned 49 clone's hole samples anti- Body positive colony hole：1H2,11F10,14F8,14H4, take one of elisa plate (4^#Plate) exemplified by, its test data is shown in Table 5, Its result that develops the color is as shown in Figure 3.

Table 5.4^#The corresponding indirect competitive ELISA detection hybridization signal (OD of plate₄₉₀Value) data analysis table

As the table shows：For 7^#、11^#With 12^#For supernatant sample, the stoste and dilution of uncontested antigen treatment group Hybridization signal is overflow, illustrates that these three sample standard deviations have very strong hybridization signal；Compete antigen treatment group stoste with Dilution hybridization signal intensities have reduction (representative has suppression valency), and the dilution group drop-out value of these three samples in various degree Become apparent from, its value is below 1, illustrate to be implicitly present in anti-free Enrofloxacin specific antibody in these three samples, answer the interpretation to be Positive colony.12^#The anti-Enrofloxacin specificity of sample is most strong, and (stoste and the signal intensity of dilution drop to by OVERFLOW Background values close to 0.3).Consult and determine that three clones are 11F10,14F8,14H4 after detection order table.

For 2^#For sample, two groups of control (OD in stoste₄₉₀Being dropped to 0.233) by 0.547 has certain suppression valency, But signal is weaker, illustrate that the ability of the clones secrete antibody is extremely unstable, it is difficult to the stable strain of monoclonal antibody is subcloned out again, because This is rejected.For 8^#For sample, although not adding the very strong (stoste of two groups of processing hybridization signals of competition antigen (overflow) dilution (3.2) have very strong hybridization signal), but with dilution factor plus competition antigen treatment group hybridization signal not Weaken, it is false positive without specific reaction relative to Enrofloxacin epitope to illustrate the hybridization signal, should give rejecting.

3. the subclone of hybridoma positive colony：

The above-mentioned positive hole detected is subcloned using limiting dilution assay, growth selection is in good condition within 10~15 days Single cell clone growth hole supernatant makees primary antibody sample to be tested and does above-mentioned two steps ELISA detection, and with above-mentioned index analysis positive rate, Treat positive rate up to 100%.The good clone of 2 hole antibody strong positives, cell growth is selected again and carries out amplification cultivation, is frozen, conservation.Figure 4 be the competitive ELISA the qualitative analysis for taking the single cell clone culture supernatant of the wheel subclone of wherein 14H4 strains second to make primary antibody, Sample loading alternative is：

The dilution of A, C, E, G column 1/10 is without ENR

The dilution ENR containing 1000ng/ml of B, D, F, H column 1/10

The plate, which has altogether, have detected 23 samples, and first sample detection zone is：(1-2)×(A-B)；By that analogy second 13 sample detection zones are：(11-12) × (G-H), wherein (1-2) × (C-D) is missing inspection hole.Can be substantially from the figure Going out A, C, E, G column has Strong positive signals, and B, D, F, H column signal of same column can be effectively suppressed, this explanation 14H4 strain two 23 single cell clone culture supernatants of wheel subclone are the positive, and positive rate reaches that 100%, 14H4 plants of subclones are obtained into Work(, and finally it is named as 14H4A1.Here it should be noted that：A represent the clone first round be subcloned after two plants of cells taking simultaneously Numbering be A A strain clones in B plants, 1 represent two plants of cells that A plants of cells are further taken after subclone and number as 1 in 2 plants No. 1 strain.

4. two strain of hybridoma 14H4A1 and 14F8B1 culture supernatants are to Enrofloxacin suppression curve：

The stability for confirming two plants of cell secretory antibodies is tested (after cell continuous passage and cryopreservation resuscitation in chessboard ELISA Cell secretory antibody titre is unaffected) after, then with the OD in above-mentioned chessboard ELISA experiments₄₉₀For 2.0~2.5 concentration points： I.e. envelope antigen is 300ng/ml, 14H4A1 supernatants 1:1600 dilutions；14F8B1 supernatants 1:800 dilutions are made as working concentration Competitive ELISA experiment determines this two plants of cells and supernatants to Enrofloxacin suppression curve, and its suppression curve is as shown in Figure 5.

Suppression curve in Fig. 5, cell line 14H4A1 IC is calculated through insertion₂₀For 0.076ng/ml, IC₅₀For 0.299ng/ml, IC₈₀For 1.579ng/ml；Cell line 14F8B1 IC₂₀For 0.171ng/ml, IC₅₀For 0.897ng/ Ml, IC₈₀For 4.637ng/ml.

Example IV：The stability and titre and affinity analysis of positive hybridoma cell strain secretory antibody.

Two plants of cells after two wheels subclone are obtained from embodiment three, take primary cell strain to expand on culture, cell respectively It is clear in liquid nitrogen cryopreservation three months；It continuous will pass after 10 generations culture supernatant and make parallel primary antibody with freezing trimestral supernatant and treat test sample This, and compare its antibody titer and to the affine of Enrofloxacin by above-mentioned antiserum indirect ELISA and indirect competitive ELISA method Power.But difference is, primary antibody dilution be not added with cBSA close in advance beyond Enrofloxacin epitope epitope (pass through The specificity of monoclonal antibody is very strong after a few wheel screenings, does not produce immunological cross-reaction with other epitopes).Meanwhile, The primary antibody dilution factor for meeting ELISA is respectively 1:100、1:200、1:400、1:800、1:1600；Primary antibody negative control is 1:400 Sp2/0Ag14 cells and supernatants.Above-mentioned two plants of cells are submitted China typical culture collection center conservation by detection after terminating Put on record.

Two strain of hybridoma (14H4A1 and 14F8B1) are as follows with titre results to envelope antigen specificity：14H4A1, 14F8B1, Sp2/0Ag14 (negative control of antibody) are respectively at after exponential phase, are continued to cultivate 24h, are made its metabolite Fully accumulation.12000r/min at supernatant, 4 DEG C is taken to centrifuge after 30min after taking its nutrient solution, 1000r/min centrifugations 10min respectively Supernatant is taken, makees primary antibody to be measured and carries out chessboard ELISA experiments.In two plants of hybridoma culture supernatant samples are tested through chessboard ELISA Hybridization signal intensities OD₄₉₀For 2.0~2.5 when, the dilution factor of envelope antigen concentration and primary antibody to be measured is shown in Table 6.

The hybridization signal intensities OD of table 6.₄₉₀For 2.0~2.5 when envelope antigen concentration and primary antibody to be measured dilution factor

Additional information：In the chessboard ELISA experiments of two strain of hybridoma signal intensity with the concentration of envelope antigen and The dilution factor of primary antibody is in concentration dependent；1:400 Sp2/0Ag14 culture supernatants are with series concentration envelope antigen without obvious Hybridization signal (OD₄₉₀0.3) value is respectively less than；Each dilution series supernatant sample of two strain of hybridoma and 3 μ g/ml cBSA are coated with Hole (envelope antigen negative control) is without obvious hybridization signal (OD₄₉₀0.2) value is respectively less than.

Embodiment five：Positive hybridoma cell strain secretory antibody is analyzed Enrofloxacin specificity.

The hole (100 μ l/ holes) on A-G columns in elisa plate is coated with 300ng/ml ENR-cBSA, with 300ng/mlcBSA bags By the hole (100 μ l/ holes) on H columns in elisa plate, eight pieces of elisa plates are coated with altogether, coating-washing-closing-washs that (method is same again On) standby afterwards.14H4A1/14F8B1 cells and supernatants are pressed 1 respectively using 0.2%OVA/PBS liquid:800 and 1:400 It is standby that dilution proportion makees work primary antibody.It is respectively that Enrofloxacin, Ciprofloxacin, promise fluorine is husky with 0.01M PBS liquid (pH=7.4) again Star, Ofloxacin are diluted to 0.2,1,5,25,125, six dilution factors such as 625ng/ml, it is control to concurrently set PBS liquid, above-mentioned Each dilution factor sample is duplicate, makees competition antigen sample after processing standby.

The above-mentioned two parts samples with processing：Portion plus isometric 14H4A1 strains dilution supernatant, portion adds isometric 14F8B1 plants of dilution supernatants, are placed in decolorization swinging table oscillation incubation (50~100rpm) 1h after adding, multiple with each processing six The mode in hole is added in the hole on above-mentioned elisa plate A-F columns (100 μ l/ holes), elisa plate G in H columns plus two kinds of primary antibodies, PBS processing Group is compared.Primary antibody incubation-washing-secondary antibody incubation-washing-coloration method is ibid.

Tested by the Competitive assays of embodiment three, and by the 503nhibiting concentration of the various medicines of its data processing method acquisition IC₅₀, then by IC₅₀(Enrofloxacin)/IC₅₀(Enrofloxacin analog) formula calculates its coefficient correlation CR, i.e. immunological cross rate (%), it is the results detailed in Table 7.As a result show, positive hybridoma cell strain secretory antibody is very strong to Enrofloxacin specificity.

The positive hybridoma cell strain secretory antibody of table 7. is to Enrofloxacin specificity

Embodiment six：A large amount of preparations of monoclonal antibody and analysis.

With antibody titer and to the affine masterpiece judging quota of Enrofloxacin, by primary cell mass propgation supernatant with freezing Recovery and continuous passage processing culture supernatant sample are compared, it was demonstrated that above-mentioned two plants of cell secretory antibodies have very strong stabilization Property after, using inducing ascites mode in vivo and preparing a large amount of monoclonal antibody proteins, i.e., by 0.5ml pristone (norphytane)/only Dosage abdominal cavity injection Balb/c mouse, sensitization is after 7 days, by 1~2 × 10⁶Individual hybridoma/only dosage abdominal cavity injection it is above-mentioned Sensitization mouse, gathers ascites after 7~10 days, 1000rpm centrifugation 10min, to remove cell in ascites, take after supernatant 12000rpm from Heart 30min, to remove cell fragment in ascites, then takes containing a large amount of enrofloxacin monoclonal antibody albumen in supernatant, the supernatant, Survey OD₂₈₀With OD₂₆₀The preliminary demarcation total protein content of value, takes a part to carry out indirect ELISA and indirect competitive ELISA experiment, point Analyse sample in antibody titer and to Enrofloxacin affinity (the same culture supernatant of chessboard elisa assay method, unlike abdomen The dilution factor that water makees primary antibody to be measured is respectively 1:10000、1:30000、1:90000、1:270000、1:810000), remainder Packing freezes standby after -80 DEG C.

After confirming that two plants of cells and supernatants have very strong affinity to Enrofloxacin, in order to explore two plants of monoclonal antibody reality Application feasibility, using luring method largely to produce monoclonal antibody albumen in vivo, above-mentioned two plants of cells have planted 6 Balb/C mouse, 7 respectively It can receive within~10 days and largely induce ascites, ascites reaches 3.5ml/ only, homophyletic is induced after ascites merging, with chessboard ELISA preruns The OD tested₄₉₀For 2.0-2.5 concentration points：I.e. envelope antigen is 300ng/ml, 14H4A1 ascites 1:160000 dilutions；14F8B1 abdomens Water 1:75000 dilutions are as working concentration, and in above-mentioned same competitive ELISA method, (it is anti-that only 14F8B1 plants of processing improves 3 times of competitions Original content) analyze its affinity to Enrofloxacin.

Competitive ELISA experiment determines this two plants of cryopreservations and continuous passage culture supernatant and Enrofloxacin is suppressed Curve, its suppression curve is as shown in Figure 6.Suppression curve in Fig. 6, cryopreservation resuscitation is calculated and continuous through insertion Passage cell strain 14H4A1 IC₂₀For 0.071ng/ml, IC₅₀For 0.236ng/ml, IC₈₀For 0.85ng/ml；Cell line 14F8B1 IC₂₀For 0.23ng/ml, IC₅₀For 0.75ng/ml, IC₈₀For 2.69ng/ml.Compare the two culture supernatant with inducing Ascites detection sensitivity (IC₅₀) do not decline, and the test limit (IC of 14F8B1 ascites₂₀) be declined slightly, the accuracy of detection of ascites It is higher.

Compared with vitro culture supernatant, two plants of cells induce ascites antibody titre and improve 100 times or so, and the result is two The practical application of strain cell provides reliable test data.

Embodiment seven：The purifying and analysis of monoclonal antibody protein.

Ascites is diluted into 4 times of (a+three parts of 0.01M of ascites with the 0.01M PBS liquid (pH=7.4) of ice-water bath precooling PBS liquid) after, as preparing, liquid is standby.The amount of 1ml protein G resins is taken by every 10mg ascites total protein, resin is filled in antibody In albumen affinity column bed, chromatographic column is balanced with the precooling 0.01MPBS liquid (pH=7.4) of 50 times of bed volumes, at room temperature Preparation liquid is set to be filtered five times through protein G resin post, it is special to make the monoclonal antibody protein in ascites fully be carried out with Protein G Property combine, then chromatographic column is fully washed with the precooling 0.01M PBS liquid (pH=7.4) of 50 times of bed volumes, to remove adhesion Foreign protein in chromatographic column.

It is each into 1.5ml EP pipes in advance to add 100 μ l 1M Tris-Cl (pH=9.6), then to the layer through above-mentioned processing Frozen water precooling 0.1M Gly-HCl (pH=2.7) are added in analysis post to be eluted, filtrate presses the dosage fraction collection of 0.9ml/ pipes In the EP pipes of above-mentioned processing, a little filtrate measurement OD is often taken in pipe₂₈₀Protein peak, merges protein peak pipe, and amalgamation liquid is placed in In bag filter, the dialysed overnight in ice-water bath 0.01M PBS liquid (pH=7.4).Take the treatment fluid a little, a part carries out SDS- PAGE electroresis appraisals, another part carries out the indirect competitive ELISA analysis of indirect ELISA and free Enrofloxacin competition antigen, - 80 DEG C of remainder packing freezes standby.

There is very high antibody titer in confirming that two plants of cells induce ascites sample and have very to Enrofloxacin haptens On the basis of strong affinity, in order to exclude other foreign proteins in above-mentioned sample to the interference of monoclonal antibody protein (mainly pair The degradation of protease in the specific interference of antibody and ascites to monoclonal antibody) and antibody protein long-term guarantor The adverse effect deposited, by monoclonal antibody-purified to antibody specificity and to the further quantitative analysis of affinity of antibody, Ascites is induced using two plants of cells make sample and purified, with 1.5ml ascites samples, through the step affinity chromatography of Protein G one, each Sample standard deviation obtains 3ml purified monoclonal antibodies protein sample (2mg/ml).

, it is apparent that collecting pipe 3 from Fig. 7^#-5^#In contain high concentration monoclonal antibody albumen, its purity be more than or equal to 99% (only having heavy chain of antibody and the band of light chain two in electrophoretic analysis figure), further to carry out Enrofloxacin monoclonal antibody, other exempt from the result Epidemiology is tested and real application research provides sufficient antibody sample.

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Claims (3)

1. hybridoma cell strain 14H4A1 or 14F8B1,14H4A1 that secretion produces enrofloxacin monoclonal antibody are mouse hybrids Oncocyte system CCTCC NO.C201487,14F8B1 are mouse hybridoma cell system CCTCC NO.C201488.

2. the enrofloxacin monoclonal antibody of hybridoma cell strain 14H4A1 according to claim 1 or 14F8B1 secretions 14H4A1 ' or 14F8B1 '.

3. enrofloxacin monoclonal antibody 14H4A1 ' or 14F8B1 ' according to claim 2 is being prepared for detecting grace promise Purposes in Sha Xing reagent.