CN107255716A - The monoclonal antibody and its enzyme-linked immune detection method of a kind of tenuazonic acid - Google Patents

Abstract

The invention discloses a kind of monoclonal antibody of energy specific recognition tenuazonic acid and its enzyme-linked immune detection method.The method comprises the steps of firstly, preparing haptens（TEA‑CMO）, and hemocyanin is coupled respectively（KLH）, bovine serum albumin(BSA)（BSA）Obtain immunogene（TEA‑CMO‑KLH）And coating antigen（TEA‑CMO‑BSA）；Then the monoclonal antibody of tenuazonic acid is obtained by animal immune and hybridoma technology, it is a kind of quick determination method of tenuazonic acid residual, this method detection limit so as to set up indirect competitive enzyme-linked immunosorbent detection method with the tenuazonic acid monoclonal antibody（IC₁₀）For 1.00 ng/mL, IC₅₀=18.50 ng/mL, linear detection range is 3.56~96.24 ng/mL, it is possible to achieve the direct detection of tenuazonic acid, and with the mould equal no cross reaction of toxin structure analog of remaining 9 kinds of rod method, method specificity is good, and sensitivity is high.

Description

The monoclonal antibody and its enzyme-linked immune detection method of a kind of tenuazonic acid

Technical field

The invention belongs to toxin detection technology field.It is anti-more particularly, to a kind of monoclonal of tenuazonic acid Body and its enzyme-linked immune detection method.

Background technology

Tenuazonic acid (Tenuazonic Acid, TEA) is one of mould main toxic metabolic products of rod method, Its molecular structural formula is C₉H₁₃O₃N (as shown in table 2), molecular weight is 197.TEA is by Food and Drug Administration (Food and Drug Administration, FDA) poisonous chemical is included in, it is beautiful in 1979 as the mould toxin of most important rod method State's National Institute and health tissues be included in toxic chemical substance liber (《Registry of Toxic Effects of Chemical Substance》) in (Yang Xin, 2000).The mould toxin of rod method worldwide causes industrial crops and food Extensive pollution, wherein TEA muskmelon, apple, apple products, orange, olive, green pepper, pimiento, tomato, tomato product, Pollute higher in the food such as sunflower seeds, sorghum, wheat, edible oil (including olive oil, rape seed oil, sesame oil, sunflower oil).At present It is known to have more than the 70 kinds of mould toxin of rod method that there is overt toxicity, and TEA is wherein unique nitrogenous metabolite, toxicity occupies chain lattice First of the mould toxin of spore.Also, there are some researches show TEA has a variety of toxicity, TEA has carried out huge potential prestige to mankind's health care belt The side of body.

Therefore, quick, easy, sensitive tenuazonic acid detection method is set up particularly important.

At present, the detection to TEA is main based on instrumental method, and the degree of accuracy and sensitivity of instrumental method are all higher, are one Plant reliable detection method.But instrument detection needs to expend the more time, operating process is comparatively laborious outer, in addition it is also necessary to larger Fund input, therefore more difficult live screening requirements for meeting batch samples.

In addition, the report at present on TEA quick detections is also few, and detection object is TEA derivative mostly, is derived Journey is comparatively laborious, and the detection band to actual sample carrys out inconvenience.

The content of the invention

The technical problem to be solved in the present invention is to overcome the defect of above-mentioned prior art and not enough there is provided a kind of alternaria The monoclonal antibody and its enzyme-linked immune detection method of bacterium ketone acid, that the enzyme-linked immune detection method has is accurate, sensitive, quick, Easy advantage, can efficient detection tenuazonic acid, the exploitation for tenuazonic acid quick detection product provides Certain theoretical direction, food-safe monitoring is significant.

It is an object of the invention to provide a kind of monoclonal antibody of tenuazonic acid.

It is a further object of the present invention to provide a kind of monoclonal antibody of tenuazonic acid in detection alternaria bacterium Application in ketone acid.

Another object of the present invention is to provide a kind of enzyme-linked immune detection method of tenuazonic acid.

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

A kind of monoclonal antibody of tenuazonic acid, is obtained with haptens TEA-CMO couplings hemocyanin KLH Product TEA-CMO-KLH as immunogene, pass through animal immune and hybridoma technology obtain tenuazonic acid monoclonal Antibody, wherein, TEA-CMO structure is as follows：

Preferably, the animal is the female Balb/C mouse at 6 monthly ages.

Preferably, the immunization method used in the monoclonal antibody is prepared：After first immunisation, spending 3 weeks exempt from for the 2nd time Epidemic disease, hereafter carried out 1 time every 2 weeks and is immunized.

It is highly preferred that the first immunisation uses isometric Freund's complete adjuvant emulsified immunogen, afterwards immune is adopted With isometric incomplete Freund's adjuvant emulsified immunogen, immunizing dose is 100 μ L.Determine antiserum within 3rd time immune latter 7th day Potency and inhibiting rate, the 4th is immune and the 5th it is immune after equally determine sero-fast potency and inhibiting rate.In cell fusion Booster immunization is wanted within first 3 days, every mouse is not added with Freund's adjuvant, the μ L immunogenes of direct injection 100.

Preferably, the hybridoma that will be enlarged by culture in the hybridoma technology injects more than 10 week old and in advance 7~ 15d is injected in the Mice Body of 500 μ L paraffin, then collects ascites, and rotating speed 12000r/min centrifugation 10min pass through octanoic acid-sulfuric acid Ammonium method purifies the monoclonal antibody for obtaining tenuazonic acid.Preferably, the haptens TEA-CMO is by following methods system It is standby to obtain：The tenuazonic acid TEA of phase homogenous quantities and the hydrochloride CMO of carboxymethyl azanol half are pressed into mass volume ratio 23 respectively ~27mg：1mL (is preferably 25mg：1~2 mL) it is dissolved in mixed solvent A, then CMO solution is added in TEA solution, mixing After uniform, 10~20h (be preferably 70 DEG C and be heated at reflux 15h) is heated at reflux in 60~80 DEG C, is dried under reduced pressure, adds 2~4mL Concentration is the HCl dissolvings of 1~3mol/L (being preferably 3.0mL 2mol/L), and dichloromethane extracts the light brown oily that is concentrated under reduced pressure to obtain Thing, dichloromethane/n-hexane reprecipitation purified oil, obtains powder TEA-CMO；The mixed solvent A be methanol, pyridine and Water by volume 1：3~5：1 (is preferably 1：4：1) ratio is obtained by mixing.

Preferably, the immunogene is prepared by following steps：

S11：Haptens is dissolved in DMF, stirring is lower to add dicyclohexylcarbodiimide and N- hydroxyls Succinimide, is stirred overnight at 4 DEG C；Centrifugation, takes supernatant to be designated as A liquid；Wherein haptens, dicyclohexylcarbodiimide, N- The mol ratio of HOSu NHS three is 1：1.2~1.5：1.2~1.5 (be preferably 1：1.5：1.5).

S12：Take carrier protein to be dissolved in 0.1mol/L PBS, be completely dissolved postscript for B liquid；Wherein carrier protein with PBS mass volume ratio is 1mg：0.1~0.2mL (is preferably 1mg：0.1mL).

S13：A drops are entered in B liquid under stirring, 4 DEG C of 10~15h of reaction (preferably 12h) have reacted rear centrifuging and taking supernatant Liquid, is then dialysed 2~4d (preferably 3d) at 4 DEG C with PBS solution, 3 dialyzates is changed daily, tenuazonic acid is obtained Immunogene.

Preferably, carrier protein described in step S12 is bovine serum albumin(BSA), human albumin, hemocyanin or egg white One kind in albumen.

It is highly preferred that the carrier protein is bovine serum albumin(BSA), hemocyanin.

Specifically, the preparation process of the monoclonal antibody of the tenuazonic acid is：

(1) mouse is immunized with immunogene TEA-CMO-KLH so that mouse there can be the antiserum of higher inhibiting rate；

(2) splenocyte and myeloma cell for taking mouse are merged, and filter out the hybridoma of energy secreting specificity antibody Cell, expands culture；

(3) it will be enlarged by the hybridoma injection Mice Body of culture, collect ascites, obtain monoclonal antibody.

It is preferred that, the mouse chosen in step (2) is that potency and inhibiting rate are high, and first three day of cell fusion will be strengthened exempting from Epidemic disease, every 500 μ l comlete antigens.

It is preferred that, screening hybridoma, probably needs 3-5 limiting dilution in step (2).

It is preferred that, in the hybridoma injection Mice Body obtained in step (3) after, to be collected by ascites, centrifugation, pure Change can just obtain monoclonal antibody.

It is preferred that, mouse is more than 10 week old, to inject within 7-15 days in advance paraffin in step (3), and dosage is 500 μ l/ Only.

It is preferred that, the centrifugal method is rotating speed 12000r/min, centrifuges 10min.Purification process uses octanoic acid-sulfuric acid Ammonium method.

In addition, said monoclonal antibody is in directly detection tenuazonic acid or prepares tenuazonic acid and directly examines Application in test agent box, also all within protection scope of the present invention.

A kind of enzyme-linked immune detection method of tenuazonic acid, the method for inspection is with above-mentioned tenuazonic acid The ELISA that monoclonal antibody is set up.

Preferably, described enzyme-linked immune detection method, specifically includes following steps：

S1. it is coated with：1mg/mL coating antigen is diluted 1000 times with coating buffer solution, and does blank control group, per hole 100 μ L, 37 DEG C of overnight incubations；

S2. wash：Incline liquid in hole, and cleaning solution is washed 2 times, is dried；

S3. close：Add 120 μ L confining liquids per hole, 37 DEG C of closing 3h dry, are inverted in 1h at 37 DEG C；

S4. it is loaded and is incubated：Standard items tenuazonic acid is dissolved in the solution that methanol is configured to 1.0mg/mL, used 0.01mol/L PBS is diluted to the μ g/L of corresponding concentration 0.00098,0.00391 μ g/L, 0.01563 μ g/L, 0.0625 μ G/L, 0.25 μ g/L, 1 μ g/L, 4 μ g/L are used as standard solution；50 μ L standard solutions and 50 μ L antiserum are added per hole (64000 times of dilution), concussion is mixed, and after 37 DEG C of reaction 40min, is washed 5 times, is dried；

Plus secondary antibody S5.：The HRP- sheep anti-mouse iggs that 100 μ L dilute 5000 times with PBS are added per hole, 37 DEG C are reacted 40min, Washing 5 times；

S6. develop the color：100 μ L nitrite ions are added per hole, then 37 DEG C of colour developing 10min add 50 μ L 10%H per hole₂SO₄ Terminating reaction；

S7. reading is determined：Light absorption value is surveyed under 450nm wavelength；Select antiserum of the absorbance in 1.0~1.5 intervals Extension rate is antiserum titre, and sero-fast effect is drawn by its inhibiting rate；

S8. calculate：The IC of suppression curve is calculated with Origin8.6 four parameter fitting modules₅₀Value.

The invention further relates to the monoclonal antibody of preparation, immunogene, coating antigen, haptens in detection tenuazonic acid In application.

Preferably, the monoclonal antibody, immunogene, coating antigen and haptens are applied to prepare tenuazonic acid Direct detection kit and colloidal gold strip in terms of.

Compared with prior art, the beneficial effects of the invention are as follows：

(1) anti-tenuazonic acid monoclonal antibody prepared by the present invention, does not intersect instead with other structures analog Should, specificity is good, and the enzyme-linked immune detection method of foundation can realize the direct detection to tenuazonic acid, convenient fast It is prompt.

(2) standard curve for the indirect enzyme-linked immunosorbent detection method that the present invention is set up, detection limit (IC₁₀) it is 1.00ng/mL, IC₅₀=18.50ng/mL, linear detection range is 3.56~96.24ng/mL.TIANZHU XINGNAO Capsul in fruit juice, beer sample Between 85.5%~119.7%, with the good (R of HPLC method testing results correlation²=0.9132), show foundation of the present invention IcELISA detection methods can apply to the detection of actual sample.

(3) present invention establishes alternaria using the monoclonal antibody of immunogene TEA-CMO-KLH preparations as coating antigen The icELISA methods of bacterium ketone acid detection, to detect that the content of tenuazonic acid in food provides detection rapidly and efficiently Means, the method is reliable and stable, and cost is relatively low, for tenuazonic acid quick detection kit, colloidal gold strip Research and development are significant.

Brief description of the drawings

Fig. 1 is tenuazonic acid artificial antigen TEA-CMO-KLH uv absorption spectra.

Fig. 2 is tenuazonic acid artificial antigen TEA-CMO-BSA uv absorption spectra.

Fig. 3 is the canonical plotting of tenuazonic acid.

Embodiment

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

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

The preparation of the tenuazonic acid immunogene of embodiment 1 and sign

1st, the preparation of tenuazonic acid haptens

By 50mg tenuazonic acids (TEA, Mw=197.1) and 50mg carboxymethyls azanol half hydrochloride (CMO, Mw= 109.3) 4.0mL, 2.0mL mixed solvent (methanol are dissolved in respectively:Pyridine:Water=1:4:1) in, then by the CMO after dissolving Solution is slowly added in the TEA solution after dissolving, after being well mixed, and is placed in oil bath pan 70 DEG C and is heated at reflux 15h.Reaction terminates (whether monitoring TEA reacts complete, and such as unreacted can continue reaction completely), mixture is dried under reduced pressure, 3.0mL is added afterwards 2mol/L HCl solution dissolving dried object.With the synthetic in the multiple aqueous phase extracted of dichloromethane, merge organic phase, depressurize dense Contract to obtain light brown grease.Purified by dichloromethane/n-hexane reprecipitation, obtain light brown powder, as haptens TEA- CMO.TEA structure is as follows：

2nd, the preparation of tenuazonic acid immunogene and coating antigen

Weigh haptens (TEA-CMO) 0.1mmoL to be dissolved in 0.5mL DMFs (DMF) solvent, stir Add 0.0309g (0.15mmoL) dicyclohexylcarbodiimide (DCC) and 0.0173g (0.15mmoL) N- hydroxysuccinimidyls acyl is sub- Amine (NHS), stirring reaction is stayed overnight at 4 DEG C, and centrifuged supernatant is is designated as A liquid.Weigh carrier protein (KLH/BSA/OVA) 0.02g is dissolved in the PBS (pH value 7.4) that 2mL concentration is 0.01 mol/L, and fully mark is liquid after dissolving.Under magnetic agitation, By A liquid being gradually dropped in B liquid slowly, 12h is reacted at 4 DEG C.Supernatant is removed in centrifugation after having reacted, and has as been coupled carrier protein Comlete antigen solution, then dialysed 3d with PBS solution at 4 DEG C, 3 dialyzates changed daily, small molecular weight impurity is removed, obtains Immunogene and coating antigen.Immunogene and coating antigen are sub-packed in 1.5mL centrifuge tubes with 1mg/mL concentration, frozen in -20 DEG C It is standby in refrigerator.

3rd, tenuazonic acid immunogene and the sign of coating antigen

(1) identification of immunogene and coating antigen is determined using the method for UV scanning in 200~400nm wave-length coverages The ultra-violet absorption spectrum of immunogene and coating antigen, compares the scanning curve of different material, and whether identification haptens and carrier protein It is coupled successfully.It is special if be coupled successfully because carrier protein and haptens all have maximum absorption band under the conditions of ultraviolet spectra Levying absworption peak can be overlapped mutually, so as to cause the blue shift of maximum absorption band.

(2) as shown in Figure 1 and Figure 2, there is carrier protein characteristic absorption peak in 280nm or so, the characteristic absorption peak of immunogene is all Obvious blue shift is occurred in that, so can prove that TEA immunogenes are synthesized successfully with coating antigen from uv scan result.

The preparation of the tenuazonic acid monoclonal antibody of embodiment 2

1st, animal immune

During the female Balb/C mouse first immunisation at 6 monthly ages, the former amount of injecting immune only, takes 1 mg/mL's for 0.1mL/ Immunogene adds isometric Freund's complete adjuvant, and the liquid after emulsification fully is in mouse back subcutaneous abdomen multi-point injection, every point About 200 μ l；Carry out being immunized for the 2nd time after 21 days, immunizing dose 0.1mL/ only, is emulsified with incomplete Freund's adjuvant；Later every 14 Its booster immunization 2 times, is typically immunized 5 times altogether.This experiment is after the 3rd time immune and the 5th is immune, and interval is taken a blood sample after 7 days and carried out Antiserum titre is determined.3 days before cell fusion, booster immunization is carried out to mouse, directly intraperitoneal injection 0.1mL immunogenes (are not added with Adjuvant).After mouse immune, carry out number of animals, management and record, note the health status of mouse.Immunization protocol is shown in Table 1 institute Show.

Table 1Balb/C mouse immunization protocols

2nd, antiserum effect analysis

The 7th day after (1) the 3rd time and the 5th booster immunization, mouse tail blood sampling 200 μ L, 37 DEG C of incubation 30min, 6000r/min centrifuges 10min, takes supernatant, plus isometric glycerine, -20 DEG C save backup.

(2) sero-fast potency and specificity are determined using indirect elisa method, its step is as follows：

S1. it is coated with：1mg/mL envelope antigen is diluted 1000 times with coating buffer solution, and does blank control group, 100 μ L/ holes are added in 96 hole elisa Plates, are placed in 37 DEG C of water baths and are incubated overnight.

S2. wash：Incline liquid in hole, and setting board-washing machine parameter adds the μ L of cleaning solution 300 per hole, and then board-washing 2 times dries Cleaning solution.

S3. close：Add 120 μ L confining liquids per hole, 37 DEG C of closing 3h dry liquid in hole, are placed in 37 DEG C of baking oven 1h to baking It is dry.

Plus antiserum and standard items S4.：By antiserum gradient dilution；Tenuazonic acid standard items are dissolved in appropriate first Alcohol is configured to 1.0mg/mL standard solution, and 4 DEG C save backup.Potency is arranged：Add 50 μ L solution dilution blanks and 50 μ L per hole The antiserum of gradient dilution, last holes adds primary antibody dilution to make blank control；Suppress row：Add 50 μ L standard solution per hole With the antiserum of 50 μ L gradient dilutions, last holes adds standard dilutions to make blank control；37 DEG C of incubation 40min, board-washing 5 It is secondary.

Plus secondary antibody S5.：100 μ L HRP- sheep anti-mouse iggs (PBS dilutes 5000 times) are added per hole, 37 DEG C of constant water bath box are anti- 40min is answered, is washed 5 times.

S6. develop the color：A liquid and B liquid in colour reagent box is mixed in equal volume, 100 μ L are added per hole, 37 DEG C of constant temperature are placed in Water bath colour developing 10min, then adds 50 μ L terminate liquid (10%H per hole₂SO₄)。

S7. reading is determined：Under the conditions of wavelength 450nm light absorption value (OD) is read with ELIASA.

(3) antiserum extension rate of the selection absorbance in 1.0~1.5 intervals is antiserum titre, sero-fast Effect is drawn by its inhibiting rate.The calculation formula of inhibiting rate is as follows：

Antibody effects can be represented by the inhibiting rate to finite concentration medicine：Under identical drug concentration, inhibiting rate is got over Height, then sensitivity of the antibody to this medicine is higher.

3rd, the screening of cell fusion and positive hybridoma

(1) recovery myeloma cell：Myeloma cell is taken out from liquid nitrogen, is put into rapidly in 37 DEG C of water-baths and thaws, melt 1000r/min centrifuges 7min after change, supernatant is handled in superclean bench, and add complete culture solution about in cell precipitation 1mL, dispels cell, is taken out and is put into after being mixed with complete medium in 9cm culture dishes with liquid-transfering gun, and is extended to 5~6 wares, its Between need to change liquid 1~2 time, when the cell of each culture dish is paved with bottom, available for cell fusion.

(2) prepared by feeder cells：1d plucks eyeball and takes blood before cell fusion, puts to death kunming mice, after by 80% alcohol Superclean bench is moved into after immersion 5min to be dissected.Careful abdominal cut, does not break abdominal muscle, then peels off skin of abdomen, exposure Abdominal muscle, peritonaeum is provoked with pincet, opens an osculum on mouse peritoneum, injects 3mL HAT complete mediums, with liquid-transfering gun come Blowback beats suction irrigation repeatedly, and will be taken out the culture medium comprising feeder cells in abdominal cavity using liquid-transfering gun, and in culture dish Culture medium mixing, such step 2~3 time are operated, to ensure to obtain enough feeder cells.

(3) prepared by splenocyte：It will repeatedly be immunized and examine the qualified Balb/c mouse orbit bloodletting of blood, collect serum, after In 80% alcohol-pickled 5min, body surface is sterilized, superclean bench is moved into and is dissected.It is general around sterile taking-up spleen, spleen White connective tissue can be adhered to, need to be rejected with scissors, is placed in after being rinsed with RPMI-1640 basal mediums stand-by in culture dish. Culture medium is drawn with disposable syringe, left hand clamps the spleen removed with tweezers, and syringe is inserted spleen by the right hand, slow note Enter culture medium, the cell in spleen is washed out.It is repeated until spleen is switched to colorless and transparent by peony, abandons spleen. The culture medium of mixing is collected into 50mL centrifuge tubes, centrifuged after sealing, the common 8min of rotating speed 1000r/min.Discarded after centrifugation Clear liquid, it is stand-by.

(4) cell fusion：The myeloma cell for removing supernatant and immune spleen cell will be centrifuged by 1:5 or so are mixed in centrifugation In pipe, 25mL basal medium is added, is centrifuged after sealing, the common 8min of rotating speed 1000r/min.Supernatant is abandoned after centrifugation, it is stand-by.Will Myeloma cell and immune spleen cell through centrifugation mixing abandon supernatant, the unnecessary culture medium that centrifuge tube mouth down is sopped up with rifle. Because the culture medium excessively remained can influence PEG action effect.By the cell of precipitation, with finger, gently bullet is loose, and centrifuge tube is put In 37 DEG C of warm water, 37 DEG C of PEG 0.8mL are preheated to pipette tips absorption, PEG is slowly added into the thin of precipitation in 1min In born of the same parents, often Jia one and drip PEG just with pipette tips gentle agitation once to be mixed, but it is firmly unsuitable too quickly, 1min is stood, it is preheated complete Culture medium, adds 10mL in 2min, with being gently mixed, adds, must not dispel along wall, makes PEG points to open.Centrifuge tube 1000r/min centrifugation 10min are sealed, after centrifugation, supernatant is outwelled, adds HAT culture mediums, with elbow straw gently imbibition tapping, Gentle agitation, takes out the HAT culture mediums in centrifuge tube and the mixing of about 75mL or so HAT fresh cultures, before being even added to In 10 pieces of 96 well culture plates containing feeder cells that one day prepares.Keep per the HAT culture mediums of addition feeder cells in hole and HAT culture volumes containing fused cell are identical, use HAT culture mediums about 24mL or so altogether per plate.

(5) screening of positive hybridoma：HAT culture mediums are used after fusion in 7~10 days, HT culture mediums is reused and changes liquid, Transition uses complete culture solution instead after one week, 14 days according to proliferative conditions.Whne cell attachment, to when accounting for plate hole 1/3, (general cell melts Close 12 days or so), the supernatant in extracting in porous culture plate detects the specific antibody in nutrient solution with indirect ELISA, Potency and the measure of inhibiting rate, selection potency height and the positive hybridoma cell strong to Drug inhibition are carried out, fusion effect is filtered out Really best positive hole, carries out mark.Aseptically, with the cell of cluster growth in the positive hole of microscope picking, move to In advance so that in 96 well culture plates of feeder cells bed board, each archioporus clones into 8 holes, treat that cell attachment covers with 1/2~1/3 hole Behind bottom, supernatant is taken, icELISA detections, equally with potency and inhibiting rate as row figureofmerit, take the powerhouse that is positive to utilize limited Dilution method is subcloned, so repeatedly 3~4 wheels (notice that culture need to be expanded by often taking turns the positive hole cell chosen, after freeze it is standby With), until per plate, each Kong Douwei is positive and potency is close with suppression after testing, so far hybridoma cell line is successfully established, and is obtained To the hybridoma cell line of the energy homogeneous antibody of stably excreting.Picking single cell clone, being detected as full positive, to be transferred to 24 holes thin Expand culture in born of the same parents' culture plate or Tissue Culture Dish, freeze in time.

A large amount of preparations of the tenuazonic acid monoclonal antibody of embodiment 3

1st, antibody-secreting is expressed

After the hybridoma cell clone of secretion monoclonal antibody specific is obtained, generally with extracorporeal culture-ing and animal body Inside induce monoclonal antibody method and largely prepare monoclonal antibody.Conventional way is：In advance more than more than ten 8 week old The intraperitoneal injection Ye Ti Shi Que of Balb/c mouse, dosage is 0.5mL/, and the intraperitoneal injection after 1~2 liang of week in mouse hybridizes Oncocyte.Mouse state is observed after inoculating cell daily, particularly since the 7th day, dispirited, abdomen occurs in the spirit of mouse Chamber can be swollen, and at this moment more pay close attention to the state of mouse, and ascites is taken with disposable syringe is sterile before dead mouse, will The ascites 12000r/min of collection centrifuges 10 min, removes upper-layer fat and lowermost fibre albumen, collects intermediate layer, uses IcELISA methods determine its potency and inhibiting rate, are saved backup at -20 DEG C.

2nd, antibody purification

The purification process that the present invention is utilized is caprylic acid-ammonium, and specific method is as follows：

(1) 4 DEG C of 12000r/min centrifugation 15min of ascites are taken, the removal of impurity is gone.

(2) ascites is taken：Acetate buffer solution by volume 1:2 mixing, are stirred at room temperature lower addition caprylic acid.Every milliliter of abdomen Water adds 33 μ L caprylic acids, adjusts pH to 4.8.

(3) mixing 30min, 4 DEG C of standing 2h are stirred at room temperature, it is fully precipitated.

(4) 4 DEG C, 12000r/min centrifugation 15min, abandon precipitation.

(5) supernatant adds the 0.1mol/L pH 7.4 of 1/10 volume after sand core funnel or 125 μm of nylon net filter PBS, adjust pH value to 7.4 with 2mol/L NaOH.

(6) in the ammonium sulfate that 0.277g/mL is added in 30min under ice bath, it is 45% to make its saturation degree.

(7) 4 DEG C of standings more than 1h, then 4 DEG C, 10000r/min centrifugation 30min, abandon supernatant.

(8) precipitation appropriate pH 7.4 PBS dissolves, 4 DEG C of dialysis 3 in 5L PBS (0.0l mol/L, pH 7.4) My god, 3 not good liquors are changed daily；Dialysis is dispensed after finishing, and rearmounted -20 DEG C save backup.

The indirect competitive enzyme-linked immunosorbent of embodiment 4 determines the foundation of tenuazonic acid method

1st, indirect competitive enzyme-linked immunosorbent (icELISA) reaction specifically includes following steps：

S1. it is coated with：Coating antigen is diluted to suitable concn with coating buffer, added in ELISA Plate hole, 100 μ L/ holes, 37 DEG C In water bath overnight.

S2. wash：Incline liquid in hole, and board-washing 2 times adds the μ L of cleaning solution 300 per hole, dries.

S3. close：120 μ L confining liquids are added per hole, 37 DEG C of closing 3h dry, are inverted in 1h in 37 DEG C of baking ovens standby.

S4. it is loaded and is incubated：Antibody and 50 μ L drug dilution liquid that 50 μ L dilute certain multiple are added per hole；Concussion is mixed It is even, reacted in 37 DEG C of water baths after 40min, board-washing machine washing plate 5 times adds the μ L of cleaning solution 250 per hole, dried.

S5. secondary antibody is added：HRP- sheep anti mouses-goat anti-rabbit igg that 100 μ L dilute 5000 times, 37 DEG C of water baths are added per hole Middle reaction 40min, rear board-washing 5 times is dried.

S6. develop the color：The μ L of nitrite ion 100 are added per hole, is placed in 37 DEG C of water baths after the 10min that develops the color, 50 μ L is added per hole Terminate liquid (10%H₂SO₄)。

S7. determine：Each hole A is determined with enzyme-linked immunosorbent assay instrument_450nmLight absorption value.

S8. calculate：The IC of suppression curve is calculated with Origin8.6 four parameter fitting modules₅₀Value.

2nd, prepare 0.00098 μ g/L, 0.00391 μ g/L, 0.01563 μ g/L, 0.0625 μ g/L, 0.25 μ g/L, 1 μ g/L, 4 μ g/L tenuazonic acid standard solution, sets up the standard curve of indirect enzyme-linked immunosorbent detection method.

Method detection is limited to (IC₁₀) it is 1.00ng/mL, IC₅₀=18.50ng/mL, linear detection range be 3.56~ 96.24ng/mL。

The measure of the antibody cross reaction rate of embodiment 5 and the measure of average recovery

1st, by the optimal envelope antigen concentration of gained and optimal antiserum extension rate in embodiment 4, with TEA, AME, AOH, ITeA, DON, AFB1, OA, OB, T-2, ZEA analogue are competition antigen, carry out indirect competitive ELISA experiment, and detection is thin The specificity of Alternariaspp ketone acid monoclonal antibody, its half-inhibition concentration (IC₅₀) and cross reacting rate (CR) numerical value in table 2 List.

The tenuazonic acid icELISA of table 2 antibody specificity (n=3)

Test result indicates that, anti-TEA antibody and the mould equal no cross reaction of toxin structure analog of remaining 9 kinds of rod method (answer IC₅₀1000ng/mL is all higher than, 0.1%) cross reacting rate is respectively less than, illustrates that the monoclonal antibody is right in actual applications Preferably, the icELISA detection methods specificity is good for TEA specificity.

2nd, average recovery is determined：

Mark-on cider, grape juice, beer juice sample carry out icELISA measure after reclaiming.It the results are shown in Table 3.

The thin Alternaria alternata bacterium ketone acid icELISA samples addition recovery experiment (n=3) of table 3

Test result indicates that, the TIANZHU XINGNAO Capsul of three kinds of samples is between 85.5%~119.7%, CV<15%, method is accurate Exactness is good, illustrates that the monoclonal antibody is applicable to the detection of TEA in actual sample with icELISA detection methods.

Claims (10)

1. a kind of monoclonal antibody of energy specific recognition tenuazonic acid, it is characterised in that be with haptens TEA-CMO The product TEA-CMO-KLH that coupling hemocyanin KLH is obtained obtains thin as immunogene by animal immune and hybridoma technology Alternariaspp ketone acid monoclonal antibody.

2. monoclonal antibody according to claim 1, it is characterised in that the haptens TEA-CMO is by following methods Prepare：The tenuazonic acid TEA of phase homogenous quantities and the hydrochloride CMO of carboxymethyl azanol half are pressed into mass volume ratio respectively 23~27 mg：1~2 mL is dissolved in mixed solvent A, then adds CMO solution in TEA solution, after being well mixed, in 60~ 80 DEG C are heated at reflux 10~20 h, are dried under reduced pressure, and add the mol/L of 2~4 mL 1~3 HCl dissolvings, and dichloromethane extraction subtracts Pressure is concentrated to give light brown grease, and dichloromethane/n-hexane reprecipitation purified oil obtains powder TEA-CMO；The mixing Solvent orange 2 A is methanol, pyridine and water by volume 1：3~5：1 ratio is obtained by mixing.

3. monoclonal antibody according to claim 1, it is characterised in that prepare the immune side used in the monoclonal antibody Method is：After first immunisation, spend 3 weeks and carry out the 2nd time and be immunized, it is hereafter immune every progress in 2 weeks 1 time.

4. monoclonal antibody according to claim 3, it is characterised in that the first immunisation is complete using isometric Freund Full adjuvant emulsion immunogene, the isometric incomplete Freund's adjuvant emulsified immunogen of immune use afterwards, immunizing dose is 100 μ L。

5. monoclonal antibody according to claim 1, it is characterised in that the animal is small for the female Balb/C at 6 monthly ages Mouse.

6. monoclonal antibody according to claim 1, it is characterised in that the immunogene is prepared into by following steps Arrive：

S11：Haptens is dissolved in DMF, stirring is lower to add dicyclohexylcarbodiimide and N- hydroxysuccinimidyls Acid imide, is stirred overnight at 4 DEG C；Centrifugation, takes supernatant to be designated as A liquid；Wherein haptens, dicyclohexylcarbodiimide, N- hydroxyls The mol ratio of succinimide three is 1：1.2~1.5：1.2~1.5；

S12：Take carrier protein to be dissolved in 0.01 mol/L PBS, be completely dissolved postscript for B liquid；Wherein carrier protein and PBS Mass volume ratio be 1 mg：0.1~0.2 mL；

S13：A drops are entered in B liquid under stirring, 4 DEG C of 10~15 h of reaction have reacted rear centrifuging and taking supernatant, then used at 4 DEG C PBS solution 2~4 d of dialysis, change 3 dialyzates, obtain the immunogene of tenuazonic acid daily.

7. monoclonal antibody according to claim 6, it is characterised in that carrier protein described in step S12 is cow's serum One kind in albumin, human albumin, hemocyanin or ovalbumin.

8. any monoclonal antibody of claim 1 to 7 is in directly detection tenuazonic acid or prepares alternaria bacterium Application in the direct detection kit of ketone acid.

9. a kind of enzyme-linked immune detection method of tenuazonic acid, it is characterised in that the method for inspection is with claim 1 The Indirect cELISA set up to the monoclonal antibody of 7 any tenuazonic acids.

10. enzyme-linked immune detection method according to claim 9, it is characterised in that comprise the following steps：

S1. it is coated with：1 mg/mL coating antigen is diluted 1000 times with coating buffer solution, and does blank control group, per the μ of hole 100 L, 37 DEG C of overnight incubations；

S2. wash：Incline liquid in hole, and cleaning solution is washed 2 times, is dried；

S3. close：Add 120 μ L confining liquids per hole, 37 DEG C of 3 h of closing dry, are inverted in 1 h at 37 DEG C；

S4. it is loaded and is incubated：Standard items tenuazonic acid is dissolved in the solution that methanol is configured to 1.0 mg/mL, with 0.01 Mol/L PBS be diluted to the μ g/L of corresponding concentration 0.00098,0.00391 μ g/L, 0.01563 μ g/L, 0.0625 μ g/L, 0.25 μ g/L, 1 μ g/L, 4 μ g/L are used as standard solution；50 μ L standard solutions and 50 are added per hole μ L dilutions 6.4 × 10³Antiserum again, concussion is mixed, and after 37 DEG C of 40 min of reaction, is washed 5 times, drying；

Plus secondary antibody S5.：The HRP- sheep anti-mouse iggs that 100 μ L dilute 5000 times with PBS are added per hole, 37 DEG C of 40 min of reaction are washed Wash 5 times；

S6. develop the color：100 μ L nitrite ions are added per hole, then 37 DEG C of 10 min of colour developing add 50 μ L 10% H per hole₂SO₄ Terminating reaction；

S7. reading is determined：Light absorption value is surveyed under 450 nm wavelength；

S8. calculate：The IC of suppression curve is calculated with Origin8.6 four parameter fitting modules₅₀Value.