CN103387532A

CN103387532A - Preparation method of anti-aristololactam FI and aristolochic acid IVa monoclonal antibody, and application of monoclonal antibody

Info

Publication number: CN103387532A
Application number: CN2012101448815A
Authority: CN
Inventors: 蔡少青; 正山征洋; 黎晓维; 森永纪; 宇都拓洋; 王璇
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2012-05-11
Filing date: 2012-05-11
Publication date: 2013-11-13

Abstract

The invention belongs to the biotechnical field and the traditional Chinese medicinal detection field, and discloses a preparation method of an anti-aristololactam FI (AL-FI) and aristolochic acid IVa (AA-IVa) monoclonal antibody, and an application of the monoclonal antibody The anti-AL-FI monoclonal antibody is prepared by synthesizing the artificial immune antigen and the coating antigen of the AL-FI through the structural reconstruction of the AL-FI, and a competitive enzyme linked immunosorbent assay method is established through the monoclonal antibody. The anti-AL-FI and AA-Iva monoclonal antibody is a first monoclonal antibody aiming at AL compounds and has a high antibody sensitivity, and the affinity constant (Ka) of the monoclonal antibody to the AL-FI is 1.941*10<9>M<-1>. The monoclonal antibody can simultaneously identify the AA-IVa, and the cross reaction rate of the monoclonal antibody is 42.27%. A process for the qualitative/quantitative analysis of the AL-FI and/or the AA-IVa having renal cytotoxicity in relevant medicinal materials and biological samples through utilizing the monoclonal antibody based enzyme linked immunosorbent assay method has the advantages of simple, sensitive and rapid sample pretreatment, environmental protection and the like.

Description

Preparation method and the application thereof of a kind of anti-aristolo-lactam FI and aristolochic acid-I IVa monoclonal antibody

Technical field

The invention belongs to biological technical field and Chinese medicine detection field, be specifically related to the preparation method of a kind of anti-aristolo-lactam FI (AL-FI) and aristolochic acid-I IVa (AA-IVa) monoclonal antibody and, based on competitive enzyme-linked immune adsorption analysis (ELISA) detection method of this monoclonal antibody, can be used for highly sensitive, the high throughput testing of medicinal material and biological sample middle kidney toxic chemical AL-FI and/or AA-IVa.

Background technology

The herbal medicine of taking aristolochic acid-I (AA) compounds or containing this compounds can cause Aristolochic acid nephropathy (AAN), has at home and abroad caused and has shown great attention to.Aristolochic acid-I be proved to be have renal toxicity, teratogenecity, carinogenicity ^[1,2], and the research in our early stage is found, the another kind of main component of Aristolochiaceae medicinal plant: aristolo-lactam (AL) also has obvious nephrocyte renal toxicity ^[3-6]The distribution range of AL compounds in vegitabilia is extensive more than the AA compounds, in 20 genus more than 60 kind of plant of annonaceae, piperaceae, Saururaceae, Menispermaceae and the Lauraceae beyond Aristolochiaceae, report is arranged all ^[4,7]The Chinese medicine that in the conventional Chinese medicine that 2010 editions pharmacopeia are recorded, report contains the AL compounds comprises: Virginia snakeroot, Northern Dutchmanspipe Vine, Herba Houttuyniae, Rhizoma Saururi (Herba Saururi), Kadsura Pepper Stem, the Bi roots of grass, pepper, Radix stephaniae tetrandrae, Common Cissampelos Herb etc. ^[4,7]We AL-FI (structure as shown in Figure 1) that studies show that in early stage has nephrocyte toxicity (IC ₅₀=94.33 μ M, MTT), report that at present the plant that contains AL-FI comprises melon leaf Virginia snakeroot, radix aristolochiae heterophyllae, Aristolochia kaempferi, line fruit Virginia snakeroot, Herba Houttuyniae, Rhizoma Saururi (Herba Saururi), Kadsura Pepper Stem, the Bi roots of grass, pepper, ovum leaf pepper, Radix stephaniae tetrandrae, Common Cissampelos Herb, Caulis et Folium piperis and Root of Oldham Fissistigma, white leaf Root of Oldham Fissistigma, many arteries and veins Root of Oldham Fissistigma and Oxymitra velutina, Pararistolochia flos-avis, Piper argyrophyllum, P.boehimerifolium, P.hamiltonii, P.longum, P.wightii ^[4,7]

In aristolochiaceae plant, the quantitative analysis method of AL compounds relates to high performance liquid chromatography (HPLC) and diode-array detector (DAD) ^[8-10], fluorimetric detector (FLD) ^[11]And mass detector (MS) ^[12]The technology such as coupling.We the early stage studies show that, although the AL compounds has distribution in various plants, but content is generally lower, therefore utilizing the total extracts of these plants to carry out HPLC analyzes often be not easy to detect this compounds in non-aristolochiaceae plant, (this is toxic reagent must first to use chloroform, must avoid using) extract and enrichment, just can carry out the HPLC-DAD qualitative analysis, carry out quantitative analysis more difficult.This situation is totally unfavorable to the raising of the quality control of relevant Chinese medicine and safety standards.The technology that the AL compounds in non-aristolochiaceae plant of having delivered at present carries out quantitative analysis only has HPLC-DAD ^[13-15], measured the content of AL-FI in Herba Houttuyniae and Rhizoma Saururi (Herba Saururi).In the medicinal plant of piperaceae such as Kadsura Pepper Stem, the Bi roots of grass and pepper, the assay of AL compounds there is no report.The problem that the HPLC detection method of AL exists is that instrument is expensive, to operator and instrument maintenance require highly, sample pre-treatments is complicated, needs to consume poisonous organic reagent, not environmental protection, also be unfavorable for carrying out Site Detection and high throughput testing.

Based on the enzyme linked immunosorbent assay analysis method (ELISA) of monoclonal antibody have that instrument is simple, the advantage such as environmental protection, sample pre-treatments are simple, (high-throughput) detection target component content of trace ingredients especially fast.In view of the AL-FI compounds has nephrocyte toxicity and the distribution range in conventional Chinese medicine is extensive, foundation is based on the immune analysis method of AL monoclonal antibody and be applied to the qualitative/quantitative analysis of this compounds, be conducive to improve the Quality Control Technology of relevant medicinal material, ensure drug safety.But the report for AL compounds monoclonal antibody is arranged not yet so far.Therefore, the preparation method of invention AL compounds monoclonal antibody and based on the analysing and detecting method tool of this antibody-like, being of great significance.The AL compounds is the immunogenic micromolecular compound of tool not, self lack can with the active function groups of the direct coupling of carrier proteins.Thereby must transform the compound structure of AL, introduce the connecting arm with active group, then itself and carrier proteins are carried out that coupling could obtain can be for immune artificial antigen.This compounds is two dimensional structure, and there is certain technical difficulty in poorly water-soluble aspect the preparation of artificial antigen.The present invention attempts preparing the monoclonal antibody of anti-AL compounds first.

We the early stage research also show, AA-IVa (structure is as shown in Figure 1) is except the plant in the Aristolochiaceae Aristolochia (Stem of Manshurian Dutchmanspipe, root of Fangchi, Slender Dutchmanspipe Root, Virginia snakeroot, Northern Dutchmanspipe Vine, Wooly Datchmanspipe Herb, Root of Kaempfer Dutchmanspipe, melon leaf Virginia snakeroot, radix aristolochiae heterophyllae, Aristolochia kaempferi etc. ^[7,9,16]) in have outside a large amount of distributions, at Asarum (Herba Asari ^[17,18], the magnificent root of Chinese wild ginger ^[16], the copper coin root of Chinese wild ginger ^[16]Deng) in also can be separated to or detect, point out us also to need to pay close attention to this compound and especially comprise distribution situation and contents level in the Asarum of conventional Chinese medicine at the Aristolochiaceae medicinal plant.In the Aristolochiaceae medicinal material, the quantitative analysis tech of AA-IVa relates to HPLC-DAD ^[8-10,17], HPLC-FLD ^[11]And HPLC-MS ^[12]These investigative techniques exist with the AL compounds and detect same problem, and namely sample pre-treatments is complicated, need to consume a large amount of organic reagents, and instrument and maintenance cost high.

Although the report of the monoclonal antibody of existing monomer about anti-AA-I and anti-AA-II so far, not yet have specially the report for the monoclonal antibody of AA-IVa.And AA-IVa also has widely and to distribute in the conventional Chinese medicine of Aristolochiaceae, therefore, the preparation method of the monoclonal antibody of the anti-AA-IVa of invention and based on the analysing and detecting method of this antibody also tool be of great significance.Southern iron in 2010 is expensive etc. ^[19]The monoclonal antibody for AA-I of report also can be identified AA-IVa simultaneously, and cross reacting rate is 31.2%.Yet the monoclonal antibody of identifying simultaneously AL compounds and AA-IVa there is not yet report.

Summary of the invention

Main purpose of the present invention is for the monoclonal antibody of a kind of anti-AL-FI and AA-IVa is provided, and a kind of ELISA test kit that detects AL-FI and/or AA-IVa.Concrete summary of the invention is as follows:

1, the invention provides AL artificial semiantigen, immunizing antigen and envelope antigen

By AL-FI is carried out structure of modification, add the 4 carbon connecting arms of Succinic anhydried introducing with carboxyl, more than 100 hours, synthesize the succinylated aristolo-lactam FI of artificial semiantigen (SAL-FI) through DMAP (DMAP) catalyzed reaction.Use again the activated carboxylic methods such as carbodiimide (EDC) and N-hydroxy-succinamide (NHS) make SAL-FI respectively with keyhole limpet hemocyanin (Keyhole limpet haemocyanin, KLH) and human serum albumin (Human serum albumin, HSA) carry out coupling, synthetic artificial immunization antigen SAL-FI-KLH and artificial envelope antigen SAL-FI-HSA.

2, the invention provides the monoclonal antibody of a kind of anti-AL-FI and AA-IVa

The preparation method of this monoclonal antibody comprises the steps:

(1) animal immune

With some of SAL-FI-KLH immunity BALB/c mouse, get the serum of the rear mouse of immunity, detect titre (light absorption value reaches the extension rate of 1.0 o'clock serum) and the competition inhibiting rate (to the inhibiting rate of 500 times of dilute serums) of the anti-AL-FI of this serum, choose both all the mouse of higher (serum titer is more than 1000, and the competition inhibiting rate of 10 μ g/mLAL-FI is more than 50%) carry out cytogamy.

(2) cytogamy and cloning

Get the splenocyte of immune mouse of above-mentioned gained and the myeloma cell (SP2/0-Ag14) of aminopterin sensitivity and carry out polyoxyethylene glycol method (PEG) cytogamy, adopt the ELISA screening positive clone, and utilizing limiting dilution assay to carry out cloning (make in experimental implementation in each holes of 96 orifice plates and only contain a hybridoma), acquisition can be secreted the hybridoma cell strain 1F9-9B-4G of anti-AL-FI monoclonal antibody.

(3) a large amount of preparations and the purifying of monoclonal antibody

Obtain monoclonal antibody by preparing in a large number monoclonal hybridoma nutrient solution supernatant, recycling Protein G affinity chromatography antagonist carries out purifying.After obtaining the monoclonal antibody 1F9-9B-4G of purifying, carry out antibody typing, antibody titer mensuration and cross reacting rate and investigate.

(4) evaluation of monoclonal antibody

By Classification Identification, we determine that the isotype of monoclonal antibody 1F9-9B-4G is IgG1 κ, to the affinity costant (Ka) of AL-FI, are 1.941 * 10 ⁹M ^-1

The specificity that adopts competitive ELISA to measure antibody reaches the cross reacting rate with its similar compound, and this antibody is mainly identified AL-FI and AA-IVa as a result, and cross reacting rate is respectively 100% and 42.27%.To other 25 compounds with similar structures comprise aristolo-lactam, two Evil A Piao are luxuriant and rich with fragrance and the identification degree lower (＜11%) of other Aristolochic Acid compounds.We name this antibody is anti-AL-FI/AA-IVa monoclonal antibody (being called for short the AL-FI/AA-IVa monoclonal antibody).

3, the invention provides the ELISA test kit of a kind of AL-FI of detection and/or AA-IVa

This test kit comprises following content:

(1) the 96 hole enzyme plates that were coated with artificial envelope antigen SAL-FI-HSA;

(2) AL-FI/AA-IVa monoclonal antibody (primary antibodie) solution;

(3) the sheep anti mouse immunoglobulin (Ig) of peroxidase (HRP) mark (two is anti-) solution;

(4) ABTS developer: contain ABTS and H ₂O ₂Citrate buffer solution (pH 4.0);

(5) enzyme plate washing lotion: the PBS buffered soln T-PBS (pH 7.4) that contains tween (Tween-20);

(6) enzyme plate confining liquid: the PBS buffered soln S-PBS (pH 7.4) that contains skim-milk.

4, the invention provides a kind of utilize described in 2 and 3 and AL-FI/AA-IVa monoclonal antibody and the test kit method of carrying out AL-FI in medicinal material and/or AA-IVa qualitative and quantitative analysis thereof

Set up competitive enzyme-linked immune analysis (ELISA) method based on the AL-FI/AA-IVa monoclonal antibody, the quantitative linearity scope that the method is measured AL-FI is 48.83～781.2ng/mL, and the quantitative linearity scope of measuring AA-IVa is 312.5～10000ng/mL.AL-FI has widely and to distribute in the conventional Chinese medicine that pharmacopeia is recorded, and has certain nephrocyte toxicity, based on the ELISA method of AL-FI/AA-IVa monoclonal antibody, the relevant quality of medicinal material that contains AL-FI is controlled and the raising tool of safety standards is of great significance.This monoclonal antibody is also identified AA-IVa (cross reacting rate is 42.27%), because AA-IVa does not distribute in the plant of non-Aristolochiaceae, so can not disturb the detection of AL-FI in the plants such as Kadsura Pepper Stem, the Bi roots of grass and pepper of the Herba Houttuyniae of Saururaceae and Rhizoma Saururi (Herba Saururi), piperaceae.Simultaneously, just because of this monoclonal antibody is also identified AA-IVa, therefore can be applicable to detect the content of AA-IVa in aristolochiaceae plant (comprising the Chinese medicine root of Chinese wild ginger important in asarum etc.).AL-FI seldom has distribution (only limiting to the melon leaf Virginia snakeroot of Aristolochia and the line fruit Virginia snakeroot of radix aristolochiae heterophyllae and line fruit Aristolochia) at aristolochiaceae plant, therefore can not disturb AA-IVa in this section plant to detect.The method has that instrument is simple, environmental protection, sample pre-treatments are simple, can carry out the advantage such as high throughput testing, AL-FI and/or the AA-IVa in the examination medicinal herbs most in use easily and quickly.

In addition, also can be applicable to the quantitative assay of the middle AL-FI of biological sample (cell, tissue or body fluid) or AA-IVa based on the ELISA method of anti-AL-FI/AA-IVa monoclonal antibody.The research in our early stage ^[20]Show, be used for ELISA method that medicinal material AA compounds detects and can be used for equally carrying out the detection by quantitative of AA and AL compounds in biological sample, and sample treatment is simple, the easy row of detection side.

Description of drawings

Fig. 1 is the chemical structure of AL-FI and AA-IVa

Fig. 2 is the synthetic route of artificial immunizing antigen SAL-FI-KLH

Fig. 3 is AL-FI concentration-absorbancy curve (typical curve of competitive ELISA)

Embodiment

The invention will be further described below in conjunction with embodiment and accompanying drawing:

Synthesizing of embodiment 1AL artificial semiantigen, immunizing antigen and envelope antigen

1, artificial semiantigen SAL-FI's is synthetic

AL-FI (＞98%) 70mg is dissolved in the 7mL pyridine, and induction stirring, to dissolving fully, adds 20 times of amount Succinic anhydrieds and 0.5 times of amount catalyzer DMAP (DMAP), and the room temperature lower magnetic force stirred 111 hours.After reaction is completed, toward drip in reaction solution 10% concentrated hydrochloric acid aqueous solution transfer pH to 3 with in and pyridine, then use ethyl acetate equal-volume extractive reaction liquid for several times, finally with pure water washing ethyl acetate layer for several times to pH be 7, the combined ethyl acetate layer also reclaims solvent, obtains thick product.Utilize preparative liquid chromatography to carry out purifying, obtain artificial semiantigen SAL-FI.

2, the preparation of artificial immunization antigen (SAL-FI-KLH)

8.2mg SAL-FI is dissolved in 1mL DMSO, under the induction stirring state, the NHS 2.5mg that will be dissolved in the EDC 5.0mg of 200 μ L MES buffered soln (pH 4.7) and be dissolved in 50 μ L DMSO adds in reaction system, is transferred to rapidly 4 ℃ of refrigeration chamber stirring reaction 15min and forms reactive intermediate with activation SAL-FI; After activation was completed, the KLH 5.0mg that will be dissolved in 200 μ L MES buffered soln (pH 4.7) under the induction stirring state dropwise joined in reaction system, continued stirring reaction 6 hours under 4 ℃; Dialyse (4 ℃) with the dialysis membrane of MWCO 6000-8000, the interval reasonable time is changed deionized water; After dialysis is completed, change the film content over to centrifuge tube ,-80 ℃ of refrigerator freezing a few hours, to take out after fully charge, lyophilize until be the fluffy powder state, obtains SAL-FI-KLH carrier proteins mixture, and synthetic route is as shown in Figure 2.

3, the preparation of artificial envelope antigen (SAL-FI-HSA)

10.0mg SAL-FI and 40.0mg EDC are dissolved in the PBS buffered soln (pH 7.4) that 2.0mL contains 20% pyridine, stir 15min and form reactive intermediate with activation SAL-FI; After activation step was completed, the 10.0mg HSA that will be dissolved in the PBS buffered soln (pH 7.4) that 500 μ L contain 20% pyridine under the induction stirring state dropwise joined in reaction system, continued stirring reaction 15 hours under 4 ℃; Dialysis and lyophilize step, with the SAL-FI-KLH part, obtain SAL-FI-HSA carrier proteins mixture.

The preparation of embodiment 2 monoclonal antibodies

1, animal immune

The 8M urea soln that will contain 5mg/mL SAL-FI-KLH dilutes 25 times with PBS, and with isopyknic Freund's complete adjuvant, is mixed into emulsion, some of abdominal injection male BALB/c mouse in 7 age in week, every 0.5mL (50 μ g).First immunisation is with same dosage booster immunization after 2 weeks, and adjuvant is replaced by Freund's incomplete adjuvant.The PBS solution (not containing adjuvant) of second immunisation pneumoretroperitoneum injection SAL-FI-KLH, every 0.5mL (100 μ g), immunity is 2 times altogether, every 2 weeks of minor tick.Detect afterwards the competition inhibiting rate of antiserum titre and AL-FI with the ELISA method for the second time with four immunity respectively, select immune effect preferably, namely serum titer is that the competition inhibiting rate of 1187,10 μ g/mL AL-FI is that 79.0% mouse is got its splenocyte (6 * 10 ⁸Individual) carry out cytogamy operation.

2, cytogamy

Myeloma cell SP2/0-Ag14 and above-mentioned splenocyte are mixed with the ratio of 1: 7,25 ℃ of centrifugal 5min, rotating speed 1000rpm, supernatant inclines; The right hand is arrested the centrifuge tube that cell mixture is housed, with bottom (part of deposition of cells) chucked 1min in the palm of the hand leftward; Draw 1.5mL PEG solution (Sigma 1300～1500), in left hand rotating centrifugal pipe, 1min, PEG is evenly added centrifuge tube along tube wall lentamente; After adding, bottom is held in palm of the hand insulation 1min, promotes reaction to carry out; Draw 13.5mL eRDF nutrient solution, uniform speed slow ground splashes in centrifuge tube along wall, continues 5min, simultaneously the rotating centrifugal pipe; With above cytogamy liquid in 25 ℃, the centrifugal 5min of revolution 1000rpm, the supernatant that inclines, then be suspended in the eRDF nutrient solution that 100mL contains 15%FBS (37 ℃ of preheatings); Amount (100～120 μ L) according to 2/hole, enter 96 porocyte culture plates (1～2/every hole) with above cell suspension inoculation, is placed in 5%CO ₂, cultivate in 37 ℃ of constant temperature cell culture incubators.

3, cell screening and cloning

Add every other day fresh HAT nutrient solution after fusion, partly changed liquid with the HAT substratum afterwards in 5 days.Observe the growing state of hybridoma every day, adopt ELISA to carry out the positive colony screening after 10 days, positive hole (absorbance＞1) is carried out amplification culture (amplifying in 24 well culture plates), carry out twice cloning with limiting dilution assay again and isolate individual cells (being that cells all in hole all comes from same parent cell), obtain the hybridoma cell line 1F9-9B-4G of secrete monoclonal antibody, the working method of limited dilution cloning is as follows:

(1) produce cell suspension from 24 well culture plates in the 15mL centrifuge tube, with 10 times of HT nutrient solution dilutions;

(2) carry out the operation such as dilution such as degree such as grade of above-mentioned cell suspension with the HT nutrient solution, dilute respectively 100,500,2500,12500,62500 and 625000 times, then the cell suspension after each group dilution is added in 96 orifice plates 2/hole (5mL transfer pipet);

(3) 5%CO ₂, cultivate in 37 ℃ of constant temperature cell culture incubators, added a HT nutrient solution, 1/hole (5mL transfer pipet) in 3～4 days;

(4) timing vision slit inner cell growing state, limiting dilution is after 7～14 days, and amplification culture is carried out in the positive hole (absorbance＞1) of finding minimum cell density level to cultivate;

Above-mentioned steps carries out twice altogether, judges whether to obtain monoclonal standard to be: in the hole of same level of density, every have the hole of cell all positive, and the cell in the hole of this level of density can be considered to obtain from the individual cells clone at this moment.And in so positive hole, the antibody that cell colony produces is monoclonal antibody.

4, a large amount of preparations of monoclonal antibody

The hybridoma cell line (1F9-9B-4G) that screens is cultivated with the eRDF nutrient solution that contains 10%FBS, amplified step by step; After obtaining a certain amount of hybridoma, be replaced with the RD-1 serum-free medium and continue to cultivate more than 14 days; Change nutrient solution over to centrifuge tube centrifugal 5min, rotating speed 1000rpm, merge supernatant; First use the glass fibre membrane filtration, after 0.45 μ m filter membrane.

5, the purifying of monoclonal antibody

Rinse Protein G affinity column, flow velocity 1mL/min with the about 20mM phosphate buffer soln of 10 times of column volumes (pH 7.0); With the serum-free medium upper prop that has filtered, flow velocity 0.5mL/min; Use phosphate buffer soln (pH 7.0) to rinse pillar to eliminate non-specific absorption, flow velocity 1mL/min, measure the absorbance (take pure water as blank) of flow point at 280nm, when A＜0.05, illustrate that impurity albumen removes substantially, stop rinsing; Carry out wash-out with the citrate buffer solution (pH 2.7) of 100mM, flow velocity 1mL/min, each stream part all uses 1M Tris buffered soln (pH 9.0) to neutralize, and measures its absorbance at 280nm; The flow point that merges all A＞0.05, neutralize with appropriate 1M Tris buffer salt solution (pH9.0); Pure water dialysis 4～5 times, dialysis membrane specification: MWCO 6000～8000; Lyophilize, the target monoclonal antibody 1F9-9B-4G of acquisition purifying.

The evaluation of embodiment 3 monoclonal antibodies

1, the purity detecting of monoclonal antibody, antibody typing and tire (Ka) measure

The monoclonal antibody 1F9-9B-4G purity of measuring foregoing invention with the sandwich ELISA method is 33.38%; Measure the specification sheets requirement of test kit and mouse source antibody κ, λ chain Rapid detection test strip by mouse source heavy chain of antibody isotype, the monoclonal antibody solution of getting purifying operates, the heavy chain type of determining monoclonal antibody 1F9-9B-4G is IgG1, and the light chain type is κ.With reference to Friguet etc. ^[21]Method carry out antibody titer and detect, recording monoclonal antibody 1F9-9B-4G is 1.941 * 10 to the affinity costant (Ka) of AL-FI ⁹M ^-1

2, antibodies specific

The specificity that adopts competitive ELISA to measure the monoclonal antibody of foregoing invention reaches the cross reaction situation with similar compound (aristolo-lactam, two Evil A Piao phenanthrene, aristolochic acid-I).Cross reacting rate with AA-IVa is determined as example: draw the concentration of testing compound AA-IVa-absorbancy curve, and with the concentration-absorbancy curve of AL-FI on same enzyme plate, compare, method of calculation are with reference to the equation of Weiler and Zenk ^[22]: at first calculate half light absorption value A, A=A ₀* 50%, A ₀It is the light absorption value in blank well (hole that does not add competing compound) for maximum light absorption value; Again A is updated in the absorbancy of AL-FI and AA-IVa-concentration regression equation, calculates respectively the concentration of the corresponding AL-FI of A and the concentration (IC of corresponding testing compound AA-IVa ₅₀); Finally both are divided by, the percentages that obtains is the cross reacting rate of this AA-IVa.The cross reacting rate of other compounds also detects after the same method and calculates, and result as shown in Table 1 and Table 2.

Monoclonal antibody 1F9-9B-4G mainly identifies AL-FI and AA-IVa, and cross reacting rate is respectively 100% and 42.27%; Lower to other 25 the compound identification degree with similar structures, cross reacting rate＜11%.

The cross reaction situation of table 1 monoclonal antibody 1F9-9B-4G to aristolo-lactam (AL) and aporphine compound

The cross reaction situation of table 2 monoclonal antibody 1F9-9B-4G to Aristolochic Acid (AA) compound

The foundation of embodiment 4AL-FI/AA-IVa competitive ELISA measuring method

1, the 50mM carbonic acid buffer salt solution 100 μ L/ holes of 2 μ g/mL SAL-FI-HSA, 37 ℃ are coated with 96 orifice plate 1 hour;

2,0.05%T-PBS cleans 96 orifice plate 3 times, 5%S-PBS 300 μ L/ holes, 4 ℃ spend the night or 37 ℃ hatch and eliminated non-specific absorption in 1 hour;

3, to contain the 10mMNaHCO of 20%MeOH ₃For AL-FI or the AA-IVa solution of solvent preparation 3125ng/mL, then wait degree to dilute 2,2 ², 2 ³, 2 ⁴, 2 ⁵, 2 ⁶, 2 ⁷Doubly;

4,0.05%T-PBS cleans 96 orifice plate 3 times, adds each concentration solution of the 3rd step preparation and the 10mM NaHCO that contains 20%MeOH ₃As blank 50 μ L/ holes, then add 500ng/mL AL-FI/AA-IVa monoclonal antibody 50 μ L/ holes, 96 orifice plate vibrator vibrations mix 1min, hatch 1 hour for 37 ℃;

5,0.05%T-PBS cleans 96 orifice plate 3 times, adds the secondary antibodies of the HRP mark of 1000 times of dilutions, and 100 μ L/ holes, hatched 1 hour for 37 ℃;

6,0.05%T-PBS cleans 96 orifice plate 3 times, adds substrate A BTS solution, 100 μ L/ holes, and 37 ℃ were reacted 20～30 minutes;

7,405nm reading, the 490nm reference, draw AL-FI or AA-IVa concentration-absorbancy curve.

As shown in Figure 3, with the typical curve that AL-FI/AA-IVa monoclonal antibody 1F9-9B-4G sets up, the quantitative linearity scope of measuring AL-FI is 48.83～781.2ng/mL, the 50% competition inhibition concentration (IC that AL-FI detects ₅₀) be 152.9ng/mL, detectability (LOD) is 24.41ng/mL.The typical curve of measuring AA-IVa is set up in utilization with method, quantitative sex-limited scope is 312.5～10000ng/mL.

In embodiment 5 medicinal herbs most in use, the ELISA of AL-FI and/or AA-IVa detects

Accurately weighed medicinal powder 0.1g, put in the 1.5mLEP pipe, adds methyl alcohol 1000 μ L, 25 ℃ of supersound extraction 30min, and centrifugal 5min (10000rpm) reclaims supernatant; Repeat methyl alcohol supersound extraction and centrifugally operated 4 times, merge the supernatant liquor of 5 times, nitrogen dries up (60 ℃), and residue, with 1000 μ L dissolve with methanol, is crossed 0.45 μ m filter membrane standby as medicinal material sample mother liquor, two parts of mother liquors of the parallel preparation of every duplicate samples.

With the methanol mother liquor of medicinal material sample 10mM NaHCO ₃Dilute 5 times, be made into the 10mM NaHCO that contains 20%MeOH ₃Solution; Dilute respectively 1～10 times according to practical situation again before mensuration, utilize the described competitive ELISA method of embodiment 4 to detect, the detected result of AL-FI is as shown in table 3, and in Herba Houttuyniae, Rhizoma Saururi (Herba Saururi), Kadsura Pepper Stem, pepper and the Bi roots of grass, the content of AL-FI is between 0.00761～0.150mg/g.Distribution situation according to AA-IVa in aristolochiaceae plant, use the content guess value of the measured AA-IVa of ELISA test kit that we invent and detection method should be between 0.01～3.36mg/g.

The ELISA measurement result of AL-FI in the medicinal herbs most in use that table 3 pharmacopeia is recorded

We the monoclonal antibody 1F9-9B-4G of invention can identify target detect antigen A L-FI (100%) and AA-IVa (42.27%), therefore the anti-AL-FI/AA-IVa monoclonal antibody of called after (being called for short the AL-FI/AA-IVa monoclonal antibody).Known AA-IVa does not distribute in non-aristolochiaceae plant, so the AL-FI/AA-IVa monoclonal antibody can not disturb the ELISA of AL-FI in the plants such as Kadsura Pepper Stem, the Bi roots of grass and pepper of the Herba Houttuyniae of Saururaceae and Rhizoma Saururi (Herba Saururi) and piperaceae to detect to the identification of AA-IVa.Equally, this monoclonal antibody also can be applicable to detect the content of AA-IVa in aristolochiaceae plant,, because in aristolochiaceae plant, AL-FI seldom has distribution, can not disturb the detection of AA-IVa.

Distribute so widely and have nephrocyte toxicity (IC in view of this compound of AL-FI has in the conventional Chinese medicine that pharmacopeia is recorded ₅₀=94.33 μ M, MTT), and AA-IVa has distribution in aristolochiaceae plant especially Asarum (as the Chinese medicine root of Chinese wild ginger commonly used etc.), based on the ELISA method of AL-FI/AA-IVa monoclonal antibody, relevant quality of medicinal material is controlled and the raising of safety standards has great importance.The advantages such as the method has that instrument is simple, environmental protection, sample pre-treatments are simple, can carry out examination to AL-FI and/or AA-IVa in relevant medicinal material easily and quickly; Also can be used for the detection of the middle AL-FI of biological sample (tissue, cell or body fluid) and/or AA-IVa.

Describe in the above embodiments and specification sheets only for preference of the present invention, be not used for limiting the present invention.Any changes and improvements of making for the present invention, all should be considered as not breaking away from the category of patent of the present invention, and these changes and improvements all fall in the claimed scope of the invention.

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Claims

1. the artificial semiantigen of an aristolo-lactam FI (AL-FI), is characterized in that, utilizes Succinic anhydried method (by DMAP catalysis) to introduce connecting arm with carboxyl in the hydroxyl of AL-FI, and its structural formula is as follows:

。

2. the artificial immunization antigen of an AL-FI, it is characterized in that, utilize the method for carbodiimide (EDC) and N-hydroxy-succinamide (NHS) mixed catalytic, artificial semiantigen claimed in claim 1 and keyhole limpet hemocyanin (KLH) are carried out coupling, obtain artificial immunization antigen SAL-FI-KLH, its structural formula is as follows:

。

3. the artificial envelope antigen of an AL-FI, it is characterized in that, utilize the method for carbodiimide (EDC) catalysis, artificial semiantigen claimed in claim 1 and human serum albumin (HSA) are carried out coupling, obtain artificial envelope antigen SAL-FI-HSA, its structural formula is as follows:

。

4. the preparation method of the monoclonal antibody of an anti-AL-FI, it is characterized in that, utilize artificial immunization antigen SAL-FI-KLH immune mouse claimed in claim 2, get the splenocyte of immune mouse and the myeloma cell (SP2/0-Ag14) of aminopterin sensitivity and carry out polyoxyethylene glycol method (PEG) cytogamy, obtain hybridoma cell strain 1F9-9B-4G, obtain monoclonal antibody 1F9-9B-4G by a large amount of preparation Hybridoma Cell Culture liquid supernatants, recycling Protein G affinity chromatography is carried out purifying to monoclonal antibody 1F9-9B-4G.

5. competitive enzyme-linked immune adsorption analysis (ELISA) test kit that detects AL-FI and/or AA-IVa, is characterized in that containing monoclonal antibody 1F9-9B-4G claimed in claim 4.

6. test kit as claimed in claim 5, is characterized in that, contains artificial envelope antigen SAL-FI-HSA claimed in claim 3.

7. an ELISA detection method, is characterized in that, utilizes claim 5 and 6 described test kits to carry out qualitative, the quantitative assay of AL-FI and AA-IVa in herbal medicine or vegetable material.

8. an ELISA detection method, is characterized in that, utilizes claim 5 and 6 described test kits to carry out qualitative, the quantitative assay of AL-FI in herbal medicine or vegetable material or AA-IVa.

9. an ELISA detection method, is characterized in that, utilizes claim 5 and 6 described test kits to carry out qualitative, the quantitative assay of the middle AL-FI of biological sample (cell, tissue or body fluid) or AA-IVa.