CN102977210A - Monoclonal antibody preparation method of huperzine A and enzyme-linked immune detection kit thereof - Google Patents

Abstract

The invention relates to the fields of immunochemistry and biotechnology, in particular to a monoclonal antibody preparation method of huperzine A and an enzyme-linked immune detection kit of the huperzine A. The invention aims to provide an enzyme-linked immuno sorbent assay (ELISA) detection and analytical method of content of the huperzine A. the method comprises the following steps: utilizing artificial antigen immune animals which are prepared, mixing together the skin cells of an immune mice with SP2/0 myeloma cells, then preparing a monoclonal antibody of the huperzine A, and then preparing the enzyme-linked immune detection kit of the huperzine A to detect the content of the huperzine A. The monoclonal antibody preparation method of the huperzine A and the enzyme-linked immune detection kit of the huperzine A has the advantages that specificity is strong, simplicity and convenience in sample pretreatment, sensitivity is high, and field monitoring is convenient. Instrument and equipment which are needed are simple, testing costs are low, the process is simplified, sample amount is little, and practical applicability is strong. According to the monoclonal antibody preparation method of the huperzine A and an enzyme-linked immune detection kit of the huperzine A, the need of development and utilization of traditional Chinese medicine is satisfied. Further, the need of the content of the huperzine A in relevant plant bodies in the process that relevant research units detect the plants in batch is achieved.

Description

The method for preparing monoclonal antibody of selagine and enzyme-linked immunologic detecting kit thereof

Technical field

The present invention relates to immunochemistry and analyze biological technical field, relate in particular to method for preparing monoclonal antibody and the enzyme-linked immunologic detecting kit thereof of selagine.

Background technology

Selagine (huperzine A, HupA) is a kind of novel lycopsida natural alkaloid that extracts from the phenol part of Huperziaceae plants of Huperzia Herba Lycopodii serrati [Huperzinaserrata (Thumb.) Trev].As s-generation acetylcholinesterase (acetylcholinesterase, AChE) inhibitor, have the characteristics such as efficient, highly selective, reversibility.Selagine is mainly used in the treatment of alzheimer disease (alzheimer's disease, AD) at present, can significantly improve memory function damage and behavior and mood disorder.Chinese scholars studies confirm that selagine has certain therapeutic action to patients such as vascular dementia (vascular dementia, VD), mental retardation, schizophrenia, myasthenia gravis.

Plants of Huperzia is with the quantitative target of selagine as medicinal ingredients, the most frequently used detection method has thin layer chromatography scanning (TLCS) at present, ultraviolet spectrophotometry (UV), nuclear magnetic resonance spectroscopy(NMR spectroscopy) (NMR), high performance liquid chromatography (HPLC) and coupling technique thereof etc., require high but exist to laboratory and instrumentation degree, with composition to be measured separation and Extraction and to remove the pretreatment process that coexistent impurity disturbs very complicated from sample, need through extracting, separate, the sample pre-treatments of purifying and complexity such as deriving, and during operational cost, analysis ingredient is expensive, be unfavorable for promoting the use of, be difficult to adapt to rapid screening and the detection of batch sample.Thereby to seek a kind of quick, easy, sensitive and economical and practical analytical procedure be the direction of current research.

Immunoassay is to utilize the specific binding reaction of antigen and antibody for the analytical technology on basis, take antibody as core reagent, has highly selective, highly sensitive, stability by force and simple and convenient economic dispatch characteristics.Invent various quick detection kit and technology maturation both at home and abroad, can be used for the detection of micro-example.At present domestic and international that adopt all is enzyme linked immunosorbent assay (enzyme-linked immunosorbentassay, ELISA) basically, and it has the unrivaled selectivity of conventional physical and chemical analysis technology and very high sensitivity, the analysis of suitable complex material.There is report to adopt hybridoma technology to prepare the monoclonal antibody of effective constituent in the Chinese medicine both at home and abroad, foundation is take the immune analysis method of monoclonal antibody as the basis, can be used for the traditional Chinese medicine quality control of scale operation, the biopharmaceutical analysis of active ingredient of Chinese herbs, and Identification chinese herbs medicine raw material and detect its pesticide residue situation.This technology has been applied to the Effective Component of Chinese Medicines such as Potenlini, ganoderan, saikoside, ginsenoside, becomes gradually at present an important tool of medicinal plant and traditional Chinese medicine quality control based on the enzyme-linked immunosorbent assay of antigen antibody reaction.

Summary of the invention

The enzyme-linked immunosorbent assay for measuring that the purpose of this invention is to provide a kind of selagine content detection, utilize the selagine artificial antigen immune animal of preparation, chrotoplast and the SP2/0 myeloma cell of immune mouse are merged, the monoclonal antibody for preparing anti-selagine, adopt the indirect competitive enzyme-linked immunosorbent detection kit to detect the content of selagine, method is simple.

Technical scheme of the present invention may further comprise the steps for a kind of preparation method of selagine monoclonal antibody is provided:

Step 1 utilizes glutaraldehyde method to synthesize the selagine artificial antigen, and the structure of described selagine artificial antigen is shown in structural formula 1:

In the described structural formula 1, R is BSA, OVA, KLH, HSA, and BSA is bovine serum albumin, and OVA is ovalbumin, and KLH is key hole keyhole limpet hemocyanin, and HSA is human serum albumin;

Step 2 utilizes the selagine artificial antigen of step 1 gained to prepare the selagine monoclonal antibody;

Step 21 detects it and tires with selagine artificial antigen immune mouse;

To tire high mouse boosting cell and SP2/0 myeloma cell of step 22 merged under PEG, obtains positive hybridoma cell, and adopts limiting dilution assay to carry out the subclone of positive hybridoma cell;

Step 23 adopts revulsion production selagine monoclonal antibody in the Mice Body;

Step 24 adopts sad ammonium sulfate precipitation method, immune-affinity chromatography antibody purification, detects tiring and purity of selagine monoclonal antibody.

Preferably, the preparation method of above-mentioned selagine monoclonal antibody, described step 1 is specially: with haptens selagine and glutaraldehyde stirring reaction 2h; Other gets the 0.01M pH 7.2 phosphate buffer soln PBS and the mentioned solution that contain BSA and continues to stir 2-3h; Add sodium borohydride and continue to stir 1h; Then the PBS with distilled water or 0.01M pH 7.2 dialysed 4-5 days; The centrifugal supernatant liquor that obtains namely gets immunogen; The mass ratio of described haptens selagine and BSA is 3 ︰ 8.

Preferably, the preparation method of above-mentioned selagine monoclonal antibody, described step 2 is specially:

After step 21 is immunogen and Freund's complete adjuvant or the emulsification of Freund's incomplete adjuvant equal-volume with step 1 gained selagine artificial antigen, subcutaneous, muscle multiple spot immunity Balb/c mouse, per two all booster immunizations once immune 4-6 time, detect it and tire;

To tire high mouse boosting cell and SP2/0 myeloma cell of step 22 merged under PEG, obtains positive hybridoma cell, and adopts limiting dilution assay to carry out the subclone of positive hybridoma cell;

Step 23 adopts revulsion manufacture order clonal antibody in the Mice Body, positive hybridoma cell is injected in the mouse peritoneal of abdominal injection whiteruss in advance, 7-10 days, enclosing puncture under the abdominal cavity and reclaiming ascites;

Step 24 adopts sad ammonium sulfate precipitation method, immune-affinity chromatography (protein G chromatography column) antibody purification, detects antibody titer and purity.

Another technical scheme of the present invention comprises for a kind of selagine enzyme-linked immunologic detecting kit is provided: 96 hole enzyme plates, selagine envelope antigen, coated damping fluid, confining liquid, selagine standardized solution, anti-selagine monoclonal antibody, enzyme conjugates working fluid, developer, washings, stop buffer;

The structure of described selagine envelope antigen is shown in structural formula 1:

In the described structural formula 1, R is BSA, OVA, KLH or HSA, and BSA is bovine serum albumin, and OVA is ovalbumin, and KLH is key hole keyhole limpet hemocyanin, and HAS is human serum albumin;

Anti-selagine monoclonal antibody made by the preparation method of each described selagine monoclonal antibody of claim 1-3.

Preferably, above-mentioned selagine enzyme-linked immunologic detecting kit, described coated damping fluid are the carbonate buffer solution of pH 9.6;

Described confining liquid is that mass percent is 5% skim-milk;

The sheep anti mouse diluent that described enzyme conjugates working fluid is the horseradish peroxidase mark;

Described developer comprises colour developing A liquid and colour developing B liquid, and described colour developing A liquid is tetramethyl benzidine, and colour developing B liquid is phosphoric acid-citrate buffer solution;

Described washings is the phosphoric acid salt tween damping fluid of 0.01M pH 7.2-7.4;

Described stop buffer is the sulphuric acid soln of 2M.

Beneficial effect of the present invention:

(1) antibody is practical: the selagine antibody production techniques has important use value and practical significance.High specificity, the high antibody and preparation method thereof of tiring of preparation provide safeguard for the immunoassay selagine, and also the development for selagine quick detection kit in plant and the associated sample has solved technological difficulties.

(2) antibody can be produced in a large number: with the hybridoma cell strain of stably excreting go down to posterity, frozen, recovery, thereby by a large amount of manufacture order clonal antibodies of revulsion in the Mice Body, and stable in properties, high specificity.

(3) test kit can be used for the mensuration of selagine content in the actual sample.

Antibodies specific of the present invention is strong, and sample pre-treatments is easy, and is highly sensitive, is convenient to carry out field monitoring.Required plant and instrument is simple, and testing cost is low, the time is short, process simplification, and sample size is few, and is practical, can satisfy the needs of selagine content in Chinese materia medica development and use and the related research units batch detection corresponding plants body thereof.

Description of drawings

Fig. 1: the scanning spectra of haptens selagine, carrier proteins and artificial antigen etc.;

Fig. 2: the examination criteria curve of selagine.

Embodiment

By describing technology contents of the present invention, structural attitude in detail, realized purpose and effect, below in conjunction with embodiment and cooperate that accompanying drawing is detailed to give explanation.

The invention provides a kind of selagine artificial antigen, prepare the monoclonal antibody of anti-selagine and detect selagine content in plant and the associated sample.Anti-selagine monoclonal antibody of the present invention has more efficient valency and inhibition valency.The present invention also provides the indirect competitive enzyme-linked immunosorbent detection kit that detects selagine, and this test kit mainly comprises: selagine envelope antigen, anti-selagine monoclonal antibody, ELIAS secondary antibody.

The anti-selagine monoclonal antibody of the present invention has more efficient valency and specificity, can be used in the immunological method of setting up selagine in rapid detection plant and the associated sample, described method is indirect competitive ELISA, the method is lower to the requirement of plant and instrument and personnel operation, testing cost is low, can satisfy the needs that batch samples is detected.

The prerequisite of immunoassay is immunizing antigen and detects and use antibody, according to the haptenic principle of design of small molecules, drafts the haptens that adopts the synthetic multiple outstanding molecule stereo structure feature of appropriate ways, probes into basis and the rule of molecular designing.Haptenic selagine and carrier protein couplet are consisted of envelope antigen in effective artificial antigen and the ELISA detection method.With the artificial antigen immune animal behind the purifying, by the detection of serum titer, select to tire high immune mouse spleen cell and SP2/0 myeloma cell are merged, to obtain the anti-selagine monoclonal antibody of high specificity.Thereby utilize the specific immune response qualitative, quantitative ground of antigen-antibody that the selagine in the sample is detected.

One of key of this technology is: the synthetic artificial antigen that can give prominence to target molecule specificity position, according to the molecular immune chemical basic principle, to a little less than the molecule distinguishability and the chemical bond of the micromolecular compound selagine of chemical structure complexity transform, outstanding selagine molecular specificity antigenic determinant, synthesis of coupling is than suitable artificial antigen.Another key of present technique is: produce and keep the monoclonal antibody hybridoma that combines with the artificial antigen determinant, to obtain the high monoclonal antibody of required specificity.The technology that produces at present hybridoma and monoclonal antibody is very ripe, but the antibody that produces because of different cells only from the different antigenic determinants on a part reacts, the complicacy that causes producing monoclonal antibody in view of each clone is so should carry out enough tests to the antibody of gained or the substratum, serum and the ascites that contain antibody.

Processing step:

(1) utilize glutaraldehyde method to synthesize selagine artificial antigen (immunogen and envelope antigen);

Glutaraldehyde is as the homotype bi-functional cross-linking agent, two aldehyde radicals can be respectively with haptens and carrier proteins on primary amino form SchiffShi alkali, the bridging of two molecules with five carbochains connect, the preparation artificial antigen, its reaction formula is as follows:

(2) utilize the uv scan method that artificial antigen is identified;

HupA and BSA all can produce obvious absorption peak in the ultraviolet region, and the artificial antigen after both couplings can present for both different maximum absorption bands.After HupA, BSA and HupA-BSA solution is made into finite concentration with PBS, under the 200-500nm wavelength, carry out respectively spectral scan, relatively the variation of maximum absorption band.

(3) preparation and purification of selagine monoclonal antibody:

1. animal immune

After immunogen and the emulsification of Freund's complete adjuvant (Freund's incomplete adjuvant) equal-volume, subcutaneous, muscle multiple spot immunity Balb/c mouse, per two all booster immunizations once immune 4-6 time, detect it and tire.

2. cytogamy

Tire high mouse boosting cell and SP2/0 myeloma cell are merged under PEG, obtain positive hybridoma cell.

3. positive hybridoma cell strain screening

Add mouse peritoneal feeder cell and fused cell and cultivate altogether, and screen positive strain.

4. limiting dilution assay cloning screening

Utilize limiting dilution assay that cloning is carried out in positive hole, until obtain the hybridoma cell strain of the anti-selagine antibody of energy stably excreting.

5. the production of monoclonal antibody and purifying

Adopt revulsion manufacture order clonal antibody in the Mice Body, hybridoma is injected in the mouse peritoneal of abdominal injection whiteruss in advance, enclosing puncture under the 7-10 days pneumoretroperitoneums and reclaiming ascites.Adopt sad ammonium sulfate precipitation method, immune-affinity chromatography (protein G chromatography column) antibody purification, detect antibody titer and purity.

(4) composition of selagine indirect competitive enzyme-linked immunosorbent detection kit:

1. the 96 hole enzyme plates that were coated with the damping fluid of envelope antigen HupA-OVA, 1;

2. the selagine concentration of standard solution be respectively 5,10,50,100,500,1000,2500ng/mL, 6mL/ bottle, each 1 bottle;

3. selagine monoclonal antibody: contain the antibody of 0.1-1.0 μ g/mL, 6-10mL/ bottle, 1 bottle;

4. enzyme conjugates working fluid: the sheep anti mouse diluent of horseradish peroxidase mark, 10mL/ bottle, 1 bottle;

5. TMB developer: colour developing A, B liquid, 6-10mL/ bottle, each 1 bottle;

6. stop buffer: concentration is the H of 2M ₂SO ₄, 6-10mL/ bottle, 1 bottle;

7. washings: concentration is the PBST(phosphoric acid salt-tween damping fluid of 0.01M pH 7.2-7.4), 200mL/ bottle, 1 bottle.

(5) selagine content in the indirect competitive enzyme-linked immunosorbent detection kit test sample:

The selagine indirect competitive enzyme-linked immunosorbent detection kit of above-mentioned preparation can be used for the mensuration of selagine content in the actual sample.

Synthetic and the evaluation of embodiment 1 selagine artificial antigen

Adopt glutaraldehyde method, selagine is connected on BSA and the OVA, synthetic artificial antigen (immunogen and envelope antigen).Identify by uv scan whether coupling is successful, measures its coupling ratio according to mole molecular ratio formula, and concrete grammar is as follows:

1.1 the selagine artificial antigen is synthetic

Take by weighing HupA 9.0mg, be dissolved in the methyl alcohol of 1mL, under agitation dropwise add the 1.8mL 2.5%GA aqueous solution, continue under the room temperature to stir 2h, be I liquid.

With 24.0mg BSA with 5mL 0.01M PBS(pH 7.2) solution under agitation dissolves preparation II liquid,

Under the magnetic agitation, II liquid is dropwise added in the I liquid, continue to stir 2.5h under the room temperature, add again the sodium borohydride aqueous solution 3ml that contains 12mg/mL, after continuing under the room temperature to stir 1h, in the dialysis tubing of packing into, to 0.01M PBS dialysis 4 days, change dialyzate every day 2 times under 4 ℃, namely get HupA-BSA.Preserve in 4 ℃ of refrigerators, for subsequent use.The preparation method of envelope antigen HupA-OVA is identical with HupA-BSA, changes carrier proteins BSA into OVA.

1.2 the evaluation of selagine artificial antigen

Adopt ultraviolet spectrophotometer that haptens selagine, carrier proteins and artificial antigen are scanned, determine whether the coupling success according to absorption peak, the results are shown in accompanying drawing Fig. 1.Scanning spectra shows that the haptens selagine is at 308nm place maximum absorption band, carrier proteins BSA and OVA have absorption peak at the 280nm place, synthetic artificial antigen HupA-BSA compares with carrier proteins BSA and haptens HupA with the UV spectrum maximum absorption band of HupA-OVA and all changes, and with carrier proteins and haptenic characteristics, illustrate that the synthetic of artificial antigen is successfully.Experimental results show that working as carrier proteins is ovalbumin, key hole keyhole limpet hemocyanin, during human serum albumin, the synthetic of artificial antigen also is successfully.

Measure respectively selagine (A), carrier proteins (B) and artificial antigen (C) respectively in the absorption value of A material and the B material maximum absorption band wavelength am of place, bm by colorimetry, i.e. AAam, AAbm, ABam, ABbm, ACam, ACbm.Calculate A material and B material respectively at mole molecular extinction value, i.e. KAam, KAbm, KBam, the KBbm of A, B material absorbing spike strong point according to formula A=KCL.Calculate as follows the mole molecular ratio (being the coupling ratio of selagine and carrier proteins) of A material and B material:

The mole molecular ratio=(ACamKBbm-ACbmKBam)/(ACbmKAam-ACamKAbm)

(annotate: am and bm are respectively A material and B material maximum absorption band wavelength)

Through calculating, coupling ratio is as follows: immunogen HupA-BSA coupling ratio is that 10.8 ︰ 1, envelope antigen HupA-OVA coupling ratio are 8.9 ︰ 1.

The selagine artificial antigen coupling ratio that this paper synthesizes is all at document [LiQX, ZhaoMS, GeeSL, et al.Development of enzyme-linked immunosorbent assays for 4-nitrophenol andsubstituted 4-nitrophenols[J] .J Agric Food Chem, within the scope of 1991,39 (9): 1685-1692] advising.

The preparation and purification of embodiment 2 selagine monoclonal antibodies

2.1 animal immune

Choose 5 of the purebred mouse of healthy cleaning level 6 week Balb/c in age, female, the about 16-18g of body weight raises and the standard test Animal House, immunity (being provided by Fujian university of TCM Experimental Animal Center) after one week of observation.

Immunogen is diluted to 500 μ g/mL with 0.01M pH PBS, by the immunizing dose of every 100 μ g, adopts the subcutaneous multi-point injections such as inguinal region, back.First immunisation is that immunogen and Freund's complete adjuvant (CFA) equal-volume mix, and later on each booster immunization is immunogen to be mixed with Freund's incomplete adjuvant (IFA) equal-volume.Immunogen and adjuvant,, drop on the water surface and do not scatter until pull is difficult by injection pipe pull repeatedly with two syringes, namely represent the emulsification success.Its immunization protocol sees Table 1.

Table 1

In a week behind each booster immunization, from eyeball venous blood collection (100-200 μ L/ only), monitor sero-fast tiring.If tire as reaching requirement, then need continue immunity; Reach requirement (1 ︰ 10 if tire ⁴) then stop immunity.

2.2 the mensuration of tiring

Tiring of antibody in the serum highlyer illustrates that then potent antibodies concentration is higher.Basic step is as follows:

(1) coated: according to character and the requirement of experiment of antigen, with the carbonate buffer solution of pH 9.6 envelope antigen being diluted is the solution of 2 μ g/mL, with 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ hatch 2h.Discard liquid in the hole, wash plate 3 times with PBST, each 3min pats dry on thieving paper.

(2) sealing: wash plate and add confining liquid (5% skim-milk) 200 μ L/ holes, 4 ℃ spend the night or 37 ℃ hatch 2h.Discard liquid in the hole, wash plate 3 times with PBST, each 3min pats dry on thieving paper.

(3) application of sample: antiserum(antisera) is diluted to different multiples with diluent, except foremost one row hole adding negative serum was organized in contrast, all the other respectively were listed as the hole adding since the antiserum(antisera) of the doubling dilution of 1 ︰ 500, and every hole adds 100 μ L, behind the vibration mixing, place 37 ℃ to hatch 1h.Discard liquid in the hole, wash plate 3 times with PBST, each 3min pats dry on thieving paper.

(4) add enzyme labelled antibody: every hole adds ELIAS secondary antibody (diluting 10000 times) the 100 μ L of fresh dilution, hatches 40min for 37 ℃, and turned letter liquid is washed plate 3 times with PBST, and each 3min pats dry on thieving paper.

(5) color reaction: add freshly prepared nitrite ion, every hole 100 μ L behind the vibration mixing, are hatched 15min for 37 ℃, close observation, and colour developing (generally can not allow blank and negative the colour developing) to a certain degree the time in time adds stop buffer.

(6) termination reaction: every hole adds the H of 50 μ L2M ₂SO ₄The solution termination reaction, the vibration mixing.

(7) result judges: on enzyme mark determinator, with the blank well zeroing, measure its OD ₄₅₀Value.The positive serum OD of antiserum titre decision principle: P ₄₅₀Value; The negative serum OD of N ₄₅₀Value.Take P/N 〉=2.1 and P-N〉0.2 antiserum(antisera) maximum dilution multiple is antiserum titre.

Behind the 4th booster immunization, mouse has obtained higher tiring, by tiring all greater than 1 ︰ 16000 that indirect ELISA detection method records.

2.3 cytogamy

With high mouse boosting cell and the SP2/0 myeloma cell ratio mixing with 5 ︰ 1-10 ︰ 1 of tiring, the centrifugal 5min of 1200rpm.Supernatant discarded is flicked the centrifuge tube bottom with forefinger, makes the cell precipitation agglomerate loose.Evenly rotate on the other hand centrifuge tube, another hand is inhaled 1mL fusogen (PEG1450), dropwise adds in 1min, places in 37 ℃ of water-baths again and reacts 1min.Add stop buffer, slow rear fast first, and stir gently cell, the centrifugal 5min of 1200rpm.Supernatant discarded adds the HAT substratum of 50mL preheating, and cell is resuspended, gently mixing.Add with the volley of rifle fire and to inoculate in advance in 96 orifice plates of feeder cell, every hole 100 μ L spread 5 blocks of plates, put cultivation in the incubator (37 ℃, 5%CO ₂).

2.4 positive hybridoma cell strain screening

Observe 96 orifice plates every day after merging, see the clone upgrowth situation, have pollution-freely, guarantee the cell normal growth.To be seen to the cloning cluster cell number reach hundreds of above after (about 7 days), liquid is changed in preparation.To merge culture supernatant extinction in the plate with the volley of rifle fire, and discard, then the fresh HT substratum of 200 μ L is added in every hole, put in the incubator and cultivate (37 ℃, 5%CO ₂).Change liquid after 2 days, change the every hole sucking-off of the supernatant in the orifice plate 100 μ L over to coated 96 good hole enzyme plate plates, and make corresponding numbering, ELISA detects in the supernatant whether specific antibody is arranged.The hole that ELISA result is positive has acellularly in the numbering vision slit on the respective panels, if cells is arranged with nutrient solution sucking-off in the hole and add the fresh HT substratum of 200 μ L, put in the incubator and to cultivate (37 ℃, 5%CO ₂).Change liquid after 2 days, the every hole sucking-off of the supernatant in the orifice plate 100 μ L change enzyme plate over to, and make corresponding numbering, and ELISA rechecks.

2.5 limiting dilution assay cloning screening

Recheck the positive monoclonal cell hole of result, prepare to carry out subclone.With liquid-transfering gun suspension is made in the piping and druming of the cell in 96 orifice plates, got 120 cells according to count results and add in the 10mL HT substratum, respectively be inoculated in 96 orifice plates of inoculating in advance feeder cell, every hole 100 μ L, perform mark on the orifice plate, put in the incubator and to cultivate (37 ℃, 5%CO ₂).

Observe 96 orifice plates every day, see the clone upgrowth situation, have pollution-freely, guarantee the cell normal growth, in 96 orifice plates clonal growth by 1/6 o'clock of full hole, mark mono-clonal and polyclone, every block of plate is selected 24 well-grown clones, numbering.

After 1 day, the every hole sucking-off of the supernatant in the orifice plate 100 μ L are changed in the enzyme plate, and make corresponding numbering, ELISA detects supernatant.The monoclonal cell that light absorption value is higher and growth conditions is good is selected in the hole that ELISA result is positive, continues subclone, repeats 3-4 time, until cell conditioned medium ELISA result is all positive in all holes, can build strain.

After building strain cell is forwarded to 24 orifice plates from 96 orifice plates, change over to after cell covers with in the T-25 square vase, change over to after cell covers with in the T-75 square vase, treat to prepare when cell state is good frozen.

2.6 the production of monoclonal antibody and purifying

Adopt and induce the standby odd contradictive hydroperitoneum of legal system in the animal body.The abdominal injection paraffin oil is to Mice Body interior (0.5mL/ only injects at least 7 days rears of paraffin oil and can be used for preparing ascites) in advance.At the bottom for the treatment of that cell is covered with bottle substantially, collecting cell when cell state is good.Hybridoma is made suspension with elbow straw piping and druming, counting, the centrifugal 5min of 1200rpm.Supernatant discarded, with a small amount of DMEM that cell is resuspended, abdominal injection enters (every injected in mice 1-2 * 10 in the paraffin mouse body ⁶Cell).

Should be noted behind the mouse inoculation hybridoma and observe the mouse state every day.(after 7 days) cervical vertebra dislocation is put to death before mouse is dying, cuts off skin with operating scissors, tears the Dermal exposure peritonaeum, cuts an osculum at peritonaeum again, with dropper with the ascites sucking-off.The centrifugal 30min of ascites 3000rpm draws supernatant, and packing is frozen in-70 ℃.

Adopt sad ammonium sulfate precipitation method, immune-affinity chromatography (protein G chromatography column) antibody purification, detect antibody titer and purity.

Embodiment 3 detects the indirect competitive enzyme-linked immunosorbent detection kit of selagine

3.1 indirect competitive enzyme-linked immunosorbent detects ratio juris

The indirect competitive enzyme-linked immunosorbent detection method is that the mixture with the preparation of micromolecular selagine and macromolecular carrier albumen coupling is adsorbed on the solid phase carrier (96 hole enzyme plate) as envelope antigen, make solid phase antigen, then add selagine to be measured, selagine in the solid phase antigen and selagine to be measured and the antibody association reaction that is at war with, if selagine content to be measured is more, the antibody that is bonded on the solid phase antigen is just fewer, otherwise the antibody that is combined in solid phase antigen is then many, two anti-(can only with the antibodies that is combined on the solid phase antigen) who adds enzyme labelling after the reaction, the free unreacted determinand of last flush away, selagine antibody and two resists, and puts reading on the microplate reader.When one timing of antibody amount, the selagine to be measured of adding is more, and the antibody of being combined with solid phase antigen is just fewer, OD ₄₅₀Value just weakens, and inhibiting rate just increases, otherwise, OD then ₄₅₀Value increases, and inhibiting rate reduces, and therefore can according to the inhibiting rate at, selagine typical curve and testing sample of known quantity, extrapolate the concentration of selagine to be measured.

3.2 determining of antigen and antibody best effort concentration

Adopt the square formation volumetry, vertically add enzyme plate behind the coated damping fluid gradient dilution envelope antigen, extension rate is respectively 1 ︰ 100,1 ︰ 200,1 ︰ 400,1 ︰ 800,1600,4 ℃ of coated spending the night of 1 ︰ 1000,1 ︰; Laterally add enzyme plate behind the PBST gradient dilution selagine antibody, extension rate is respectively 1 ︰ 1000,1 ︰ 2000,1 ︰ 3000,1 ︰ 4000,1 ︰ 5000,1 ︰ 6000,1 ︰ 7000,1 ︰ 8000, and all the other steps are with 2.2.Envelope antigen and antiserum(antisera) diluent with doubling dilution carry out ELISA detection, the OD that relatively respectively organizes after the reaction ₄₅₀Value is with OD ₄₅₀Value is the ELISA best operating condition near 1.0 o'clock envelope antigen and antibody dilution multiple.Selecting envelope antigen (1.33mg/mL) extension rate is 1200, and antibody (0.905mg/mL) extension rate is 1 ︰ 5000, and the antibody dilution multiple of indirect competitive enzyme-linked immunosorbent detection method then is 1 ︰ 3000.

3.3 the preparation of indirect competitive enzyme-linked immunosorbent detection kit agents useful for same

(1) enzyme plate preparation: envelope antigen HupA-OVA is diluted to 1-0.5 μ g/mL with coated damping fluid, joins in the micropore of enzyme plate, 100 μ L/ holes, 4 ℃ are spent the night, and the 37 ℃ of sealings in inferior daily confining liquid 200 μ L/ holes 2h discards liquid, drying, encapsulation.

(2) washings: concentration is the PBST of 0.01M pH 7.2-7.4.

(3) selagine reference liquid: selagine is first with utilizing the PBST constant volume behind an amount of dissolve with methanol, is mixed with standard substance storage liquid, then it is diluted to that concentration is respectively 5,10,50,100,500,1000,2500ng/mL;

(4) selagine monoclonal antibody: utilize washings with 3000 times of monoclonal antibody dilutions, namely contain the selagine monoclonal antibody of 0.1-1.0 μ g/mL.

(5) TMB developer:

A liquid:, take by weighing 10mg TMB and be dissolved in the 10mL dehydrated alcohol, be i.e. the TMB mother liquor of 1mg/mL; B liquid: citric acid 1.02g; Na ₂HPO ₄12H ₂O 3.68g; H ₂O ₂0.2mL; High purity water 20mL, mixing; Before use in following ratio preparation: A liquid (1mL)+B liquid (1mL)+high purity water (10 μ L), mixing, now with the current.

(6) enzyme conjugates working fluid: utilize washings that the sheep anti mouse (1mg/mL) of horseradish peroxidase mark is diluted 10000 times.

(7) H of stop buffer: 2M ₂SO ₄, be packed as the 6mL/ bottle.

3.4 the detection method of indirect competitive enzyme-linked immunosorbent detection kit

(1) application of sample: add selagine or the sample 50 μ L of serial dilution, then add certain dilution antibody 50 μ L, behind the vibration mixing, place 37 ℃ to hatch 1h.Discard liquid in the hole, wash plate 3 times with PBST, each 3min pats dry on thieving paper.

(2) add enzyme labelled antibody: every hole adds ELIAS secondary antibody (diluting 10000 times) the 50 μ L/ holes of fresh dilution, hatches 1h for 37 ℃.Discard liquid in the hole, wash plate 3 times with PBST, each 3min pats dry on thieving paper.

(3) color reaction: add freshly prepared developer, every hole 100 μ L behind the vibration mixing, are hatched 15-20min for 37 ℃.

(4) termination reaction: every hole adds the H of 50 μ L2M ₂SO ₄The solution termination reaction, the vibration mixing.

(5) interpretation of result: on enzyme mark determinator, survey light absorption value in 450nm, calculate each standard model, treat the OD of test sample and reference sample ₄₅₀Value.Set up the regression equation of the typical curve of selagine, carry out correlation analysis.

Selagine content in the embodiment 4 indirect competitive enzyme-linked immunosorbent detection kit test sample

4.1 the preparation of need testing solution

Get 20 of Huperzine-A Tablets, Tests for Uniformities, accurately weighed, porphyrize, precision takes by weighing fine powder an amount of (being equivalent to approximately selagine 50 μ g) and puts in the 50mL volumetric flask.Add 5mL methyl alcohol, the ultrasonic 30min of jolting adds the PBST constant volume, shakes up.Precision is measured 1mL and is put the 10mL volumetric flask, adds the PBST constant volume, as need testing solution.

4.2 typical curve

Take standard model concentration logarithmic value as X-coordinate, the mapping take inhibiting rate as Y-axis, this figure is linear graph, wherein, and inhibiting rate=(with reference to OD ₄₅₀-OD to be measured ₄₅₀)/with reference to OD ₄₅₀* 100%, obtain the regression equation of the typical curve of selagine.The results are shown in Figure 2, the regression equation of the typical curve of selagine is y=0.363x-0.2377, R=0.992, and its linearity range is: 5-2500ng/mL.

4.3 sensitivity

Estimate the method for ELISA reaction sensitivity, commonly used have an IC ₅₀And detectability.IC ₅₀Be from curve and find 50%OD ₄₅₀The concentration that is worth corresponding competition thing.Detectability is the corresponding concentration of X-2SD, and wherein, X is the mean value of blank OD value, and SD is standard deviation.Carry out 10 typical curve tests, get IC ₅₀Be 85.961-107.704ng/mL, detect and be limited to 4.257-6.242ng/mL.

4.4 cross reacting rate

The selagine complex structure, its analog reference substance is difficult to obtain, thus adopt itself and several frequently seen alkaloid to carry out the cross reacting rate test, thus its specificity reflected.Get respectively selagine and other alkaloid (Strychnine, napelline, Hypaconitine, tetrahydropalmatine and Berberine) standardized solution, utilize detection kit to detect, the Criterion curve is found 50%OD from curve ₄₅₀The concentration that is worth corresponding competition thing is IC ₅₀Value.According to following formula, be used for calculating HupA and other alkaloidal cross reacting rate.

Cross reacting rate CR%=IC ₅₀HupA/IC ₅₀Analogue * 100%

The results are shown in Table 2-cross reacting rate measurement result, HupA and other alkaloidal cross reacting rate illustrate that all less than 0.01% its specificity is high.

Table 2

Alkaloid	IC 50（ng/mL）	Cross reacting rate (%)
			Selagine	107.704	100%
Strychnine	>10 5	<0.01
			Napelline	>10 5	<0.01

Hypaconitine	>10 5	<0.01
			Tetrahydropalmatine	>10 5	<0.01
Berberine	>10 5	<0.01

4.5 Precision Experiment

Select 3 enzyme plates, extract 5 micropores out for every, measure the OD of the standardized solution of 50ng/mL ₄₅₀Value, its variation coefficient is respectively 1.2%, 0.9% and 1.2%.The result is within allowed band, and the result is comparatively reliable.

4.6 repeated experiment

Get 6 parts in Huperzine-A Tablets, Tests for Uniformity sample, by " a 4.1 " standby solution of below legal system, parallel 5 times of every duplicate samples, its average content is that 359.103 μ g/g and the variation coefficient are 7.1%, and the variation coefficient is within allowed band, and the result is comparatively reliable.

4.7 the mensuration of mark-on sample recovery rate

The mensuration of mark-on sample recovery rate: utilize detection kit to measure the sample of the selagine standard substance that added 80%, 100%, 120%3 concentration, each concentration is done 3 parts, parallel 3 times of every duplicate samples is by the regression equation calculation medicament contg of setting up and the interpolation rate of recovery.The rate of recovery calculation formula of mark-on sample:

Add the rate of recovery (%)=(actual measurement content-samples contg)/adding normal content * 100%

The results are shown in Table the measurement result (n=3) of 3-mark-on sample recovery rate, the method is used for the mensuration of sample selagine, and the rate of recovery is between 94.211-108.607%, and variation coefficient scope is between 2.3-4.8%.The variation coefficient is within allowed band, and method is feasible.

Table 3

Add-on (%)	Mean value (%)	The variation coefficient (%)
			80	96.727	2.3
100	101.471	4.8
			120	106.748	2.6

4.8 the detection of Huperzine-A Tablets, Tests for Uniformity

Get Huperzine-A Tablets, Tests for Uniformity by " a 4.1 " standby sample solution of below legal system, parallel survey 5 times.According to OD ₄₅₀Value, calculating Huperzine-A Tablets, Tests for Uniformity content is 358.402 μ g/g.

The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.

Claims (5)

1. the preparation method of a selagine monoclonal antibody is characterized in that, may further comprise the steps:

Step 1 utilizes glutaraldehyde method to synthesize the selagine artificial antigen, and the structure of described selagine artificial antigen is shown in structural formula 1:

In the described structural formula 1, R is BSA, OVA, KLH, HSA, and BSA is bovine serum albumin, and OVA is ovalbumin, and KLH is key hole keyhole limpet hemocyanin, and HSA is human serum albumin;

Step 2 utilizes the selagine artificial antigen of step 1 gained to prepare the selagine monoclonal antibody;

Step 21 detects it and tires with selagine artificial antigen immune mouse;

To tire high mouse boosting cell and SP2/0 myeloma cell of step 22 merged under PEG, obtains positive hybridoma cell, and adopts limiting dilution assay to carry out the subclone of positive hybridoma cell;

Step 23 adopts revulsion production selagine monoclonal antibody in the Mice Body;

Step 24 adopts sad ammonium sulfate precipitation method, immune-affinity chromatography antibody purification, detects tiring and purity of selagine monoclonal antibody.

2. the preparation method of selagine monoclonal antibody according to claim 1 is characterized in that, described step 1 is specially: with haptens selagine and glutaraldehyde stirring reaction 2h; Other gets the 0.01M pH 7.2 phosphate buffer soln PBS and the mentioned solution that contain BSA and continues to stir 2-3h; Add sodium borohydride and continue to stir 1h; Then the PBS with distilled water or 0.01M pH 7.2 dialysed 4-5 days; The centrifugal supernatant liquor that obtains namely gets immunogen; The mass ratio of described haptens selagine and BSA is 3 ︰ 8.

3. the preparation method of selagine monoclonal antibody as claimed in claim 1 is characterized in that, described step 2 is specially:

After step 21 is immunogen and Freund's complete adjuvant or the emulsification of Freund's incomplete adjuvant equal-volume with step 1 gained selagine artificial antigen, subcutaneous, muscle multiple spot immunity Balb/c mouse, per two all booster immunizations once immune 4-6 time, detect it and tire;

To tire high mouse boosting cell and SP2/0 myeloma cell of step 22 merged under PEG, obtains positive hybridoma cell, and adopts limiting dilution assay to carry out the subclone of positive hybridoma cell;

Step 23 adopts revulsion manufacture order clonal antibody in the Mice Body, positive hybridoma cell is injected in the mouse peritoneal of abdominal injection whiteruss in advance, 7-10 days, enclosing puncture under the abdominal cavity and reclaiming ascites;

Step 24 adopts sad ammonium sulfate precipitation method, immune-affinity chromatography (protein G chromatography column) antibody purification, detects antibody titer and purity.

4. selagine enzyme-linked immunologic detecting kit, it is characterized in that, comprising: 96 hole enzyme plates, selagine envelope antigen, coated damping fluid, confining liquid, selagine standardized solution, anti-selagine monoclonal antibody, enzyme conjugates working fluid, developer, washings, stop buffer;

The structure of described selagine envelope antigen is shown in structural formula 1:

In the described structural formula 1, R is BSA, OVA, KLH, HSA, and BSA is bovine serum albumin, and OVA is ovalbumin, and KLH is key hole keyhole limpet hemocyanin, and HSA is human serum albumin;

Anti-selagine monoclonal antibody made by the preparation method of each described selagine monoclonal antibody of claim 1-3.

5. selagine enzyme-linked immunologic detecting kit according to claim 4 is characterized in that,

Described coated damping fluid is the carbonate buffer solution of pH 9.6;

Described confining liquid is that mass percent is 5% skim-milk;

The sheep anti mouse diluent that described enzyme conjugates working fluid is the horseradish peroxidase mark;

Described developer comprises colour developing A liquid and colour developing B liquid, and described colour developing A liquid is tetramethyl benzidine, and colour developing B liquid is phosphoric acid-citrate buffer solution;

Described washings is the phosphoric acid salt tween damping fluid of 0.01M pH 7.2-7.4;

Described stop buffer is the sulphuric acid soln of 2M.

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