CN101392017B

CN101392017B - Isolation and purification method of main allergic protein of humulus pollen

Info

Publication number: CN101392017B
Application number: CN2007101221779A
Authority: CN
Inventors: 尹佳; 程璇
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2007-09-21
Filing date: 2007-09-21
Publication date: 2011-12-07
Anticipated expiration: 2027-09-21
Also published as: CN101392017A

Abstract

The present invention provides a method for separating and purifying the main sensitization protein of humulus pollen. The humulus pollen is taken as raw material and prepared into crude extract and then go through gel molecular exclusion chromatography and strong anion exchange chromatography to obtain the main sensitization protein. The protein can be combined with over 89 percent of the serological specificity IgE (sIgE) of patients allergic to the humulus pollen. The apparent molecular weight of the protein measured by SDS-PAGE is about 12kDa, the isoelectric point is 4.7 and the N-terminal sequence is NH4<+>-MDNPFENGMKA-COO<->. The separation and purification of the protein lay the foundation for realizing the standardization of the allergenic preparation of the protein.

Description

The separation purification method of main allergic protein of humulus pollen

Technical field

The invention belongs to biological technical field, be specifically related to the separation purification method of main allergic protein of humulus pollen.

Background technology

Pollinosis does not wait from 1%～25% at the sickness rate of countries in the world ^[1], multinomial epidemiology survey result shows that the pollinosis patient accounts for 10%～15% of whole world total population ^[2～5], but China does not still have the epidemic data of national general population's pollinosis sickness rate at present.Before 30 years partial area minority general population was done the pollinosis epidemiology survey and show that its sickness rate can reach 5% ^[6]

The eighties was passed the sensitization pollen survey and was shown that the content of pollen humuli scandentis is only second to artemisia pollen in air summer and autumn of a lot of areas by take the lead Chinese gas that hospital of national tens of family finishes of Ye Shitai, Qiao Bingshan last century ^[7]1989-1992, Yin Jia, Ye Shitai confirm in the clinical study to Beijing area pollen nine kinds of summer and autumns: pollen humuli scandentis is the major reason that causes the northern area of China pollinosis summer and autumn ^[8], simultaneously first Application mouse-anti human IgE monoclonal antibody has been set up the detection method of human serum pollen humuli scandentis specific IgE at home ^[9], and nineteen ninety-five U.S.'s transformation reactions and the clinical immunology annual meeting on be the major reason of Chinese northern area pollinosis summer and autumn to world's first report pollen humuli scandentis ^[10]

Yin Jia etc. studies show that 1120 example pollinosis patient incidences summer and autumn in recent years: the positive per-cent of pollen humuli scandentis in China pollinosis summer and autumn patient is up to 66% (739/1120), the allergic rhinitis average age of onset that China's pollen summer and autumn (wormwood artemisia and pollen humuli scandentis) brings out is 27 years old, and the average age of onset of asthma is 32 years old ^{[11,12,13,14]}And Zhao Jing etc. find in 13～14 years old childrens respiratory tract anaphylactic disease epidemiology survey of Beijing area: artemisia pollen sensitivity person is 5.7%, and pollen humuli scandentis sensitivity person is 4.2% ^[15]Infer that in view of the above the ratio of pollen humuli scandentis disease patient in China general population should surpass 5%, promptly surpass 6,500,000 people approximately and suffer from pollen humuli scandentis disease ^{[11,12,13,14]}

The humulus grass is annual voluble herb plant, the florescence 7-9 month, the fruit phase 9-10 month, be born near the limes marginis, roadside, garden of city and countryside and between the field, between chad matter sand ground and shrubbery, vitality is extremely strong, be the common weeds of China, all there is distribution each provinces and regions except that Xinjiang, Qinghai, and also there are distribution in Japan, Korea, Russia.

Pollen humuli scandentis is important sensitization pollen China's summer and autumn, can bring out serious rhinitis summer and autumn, conjunctivitis and asthma.In population of China, 15～34 years old be summer and autumn pollen bring out the age bracket occurred frequently of rhinitis, 25～44 years old is the age bracket occurred frequently of asthma, in whole pollinosis patients summer and autumn, 37% in 5 years, and 47% can develop into seasonal asthma in 9 years, because this part patient is developed to asthma by rhinitis and focuses mostly between twenty and fifty stage of 25～54, therefore and its process need experience 5～10 years, and before 34 years old, the ratio of simple rhinitis is higher among the summer and autumn pollinosis patient; The ratio of asthma increases after 35 years old, but 18% patient's rhinitis and asthma outbreak first in same year are also arranged, and part patient is very serious because of the symptoms of asthma that summer and autumn, pollen brought out, and in the seasonal asthma patient that it brings out, 77.9% needs oral suppressing panting calming medicine; Can not put down 77.7% night for sleeping in; 30.4% needs emergency treatment input aminophylline or glucocorticoid medicine treatment ^[11,12]Nearly half pollinosis patient summer and autumn might develop into seasonal allergic asthma in 9 years of morbidity first be a clinical problem that can not be ignored.The peak age of pollen hypersensitivity rhinitis and allergic asthma morbidity summer and autumn is energetic on the occasion of life, be society's stage of creating the wealth, and therefore, seeks to stop and to intervene summer and autumn pollen hypersensitivity rhinitis extremely important to opportunity and method that asthma develops.

The World Health Organization points out that in the guidance note about the anaphylactic disease immunotherapy specific active immunotherapy is present unique methods of treatment that can stop the anaphylactic disease nature process.Therefore, should begin specific active immunotherapy as early as possible, prevent that it from developing into asthma the serious Allergic Rhinitis that summer and autumn, pollen brought out; Patient to existing asthma more should actively carry out specific active immunotherapy, prevents that asthma from further increasing the weight of.

Pollen humuli scandentis anaphylactogen diagnosis and the treatment preparation rhinitis that to be diagnosis and treatment bring out because of pollen humuli scandentis and the crucial medicament of asthma, and produce with use this medicament process in need guarantee consistent, the main allergic protein content unanimity of each batch product biological value, therefore, the main allergic protein of pollen is one of core of formulating by the stdn preparation.

Main allergic protein still is the core of following synthetic pollen diagnosis and treatment preparation.

Obtain monoclonal antibody, but, determine drug dose and concentration main allergic protein of humulus pollen content in detection of drugs and the biotechnological formulation with main allergic protein preparation.

At present very limited to the research of pollen humuli scandentis allergic protein.Sun Xiuzhen etc. ^[16]Adopt methods analyst pollen humuli scandentis allergic proteins such as SDS-PAGE, immunoblotting, discovery has 5 kinds of allergic proteins at least in 10 kinds of protein ingredients that contain, molecular weight is respectively 8.0,28.8,35.1,42.0,55.0,65.0,76.2,100.5kDa, wherein the albumen of 8.0kDa can combine with sIgE in the irritated asthmatic patient serum of 84.6% pollen humuli scandentis, it is main allergic protein, though the albumen of this molecular weight ranges had not both had yet immunologic incompetence of allergenicity and the albumen content of 11.8～23kDa is preponderated, and was little with the relation of asthma and skin test positive reaction.

Park JW ^[17]Deng finding that pollen humuli scandentis contains 10,16,20,29,5 kinds of allergic protein compositions of 42kDa; Wherein the protein of 10kDa may be main allergic protein (with the positive combination rate of pollen humuli scandentis allergic disease human serum sIgE be 72%), the N terminal amino acid sequence analysis of 10kDa allergic protein is not found that there are homology in itself and the present allergic protein of identifying, and its iso-electric point is roughly pI5.1.And Korea S another one scholar Park HS ^[18]Studies show that 13,70,80 kDa albumen are main allergic proteins of pollen humuli scandentis.

Summary of the invention

The object of the present invention is to provide the separation purification method of the main allergic protein of pollen humuli scandentis.

Albumen of the present invention is that separation and purification obtains from pollen humuli scandentis, can combine with 89% above pollen humuli scandentis allergic disease human serum specific IgE, measure its apparent molecular weight by SDS-PAGE and be about 12kDa, its pI=4.7 of isoelectrofocusing measuring, its N terminal sequence of N terminal sequence analysis revealed is: NH ₄ ⁺-MDNPFENGMKA-COO ^-

The present invention with pollen humuli scandentis as starting material, the preparation protein crude extract, show that through SDS-PAGE electrophoresis and immunoblot experiment the albumen at 12kDa place is main allergic protein of humulus pollen, the positive reaction rate of itself and pollen humuli scandentis allergic disease human serum sIgE reaches 89%.The crude extract of preparation is collected the albumen that obtains 10～38kDa molecular weight region by the gel molecular exclusion chromatography, obtain 4 elution peaks by anion-exchange chromatography at last, show through electrophoretic analysis, the 4th elution peak molecular weight of albumen is 12kDa, and a series of external immunological experiments show that it really is main allergic protein of humulus pollen.Further the main allergic protein that collection is obtained carries out the N-terminal sequential analysis, and its N terminal amino acid sequence is: NH ₄ ⁺-MDNPFENGMKA-COO ^-, by retrieval, do not find same protein, show that the albumen that is obtained is a new albumen, now with this albumen called after Hum s1.

Specifically, the method for the main allergic protein matter of separation and purification humulus grass of the present invention comprises the steps:

1) preparation pollen humuli scandentis protein crude extract;

2) gel molecular exclusion chromatography;

3) reinforcing yin essence ion exchange chromatography.

Wherein step 1) is to be raw material with the pollen humuli scandentis, after the pollen humuli scandentis degreasing, uses the PBS buffer extraction, centrifugal supernatant, through saltout, redissolution, desalination, concentrate pollen humuli scandentis albumen coarse body fluid.

The packing material of the chromatography column of gel molecular exclusion chromatography step 2 wherein) can be the sephacryl gel, what present method adopted is Sephacryl 100 prepacked columns of Amersham, can use 0.05M PBS upper prop and the wash-out of pH7.2, collecting retention volume is the elution peak of 87.25 ± 0.65ml.

Wherein the packing material of the chromatography column of step 3) reinforcing yin essence ion exchange chromatography can be sepharose, what present method adopted is the HiTrap Q Sepharose FF prepacked column of Amersham, concrete grammar is in the described reinforcing yin essence ion exchange column collection liquid with the gel molecular exclusion chromatography joins balance after pre-treatment after, carry out linear gradient with the 0.02M BisTris-HCl of the pH7.0 that contains 0～0.1M NaCl concentration gradient and wash post, again with the constant post of washing of 0.02M BisTris-HCl of the pH7.0 of 0.1M NaCl, use the 0.02M BisTris-HCl wash-out of the pH7.0 of 0.1～0.2M NaCl at last, collect elution peak.

The present invention also further with the albumen that obtains as immunogen, immune BALB/c mouse, extracting spleen cell merges with the myeloma cell, screening at last obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.

By the above-mentioned monoclonal antibody that obtains, can also further make up immunity detection reagent.

The stdn of pollen humuli scandentis allergen preparation is being prerequisite to the deep understanding of allergic protein component molecular level wherein, the separation and purification of main allergic protein of humulus pollen is laid a good foundation for the realization of its allergen standard preparationization, and its purposes and meaning show following several aspect:

1) with this as natural standard substance, be used for the foundation of the main allergic protein quantivative approach of pollen humuli scandentis allergen standard preparationization, thereby the quantitative sign that helps in the pollen humuli scandentis allergen preparation main allergic protein, and the content of main allergic protein weighed difference between each batch allergen preparation as an important parameter;

2) with the biomarker of this main allergic protein, and develop in the corresponding body/external specific diagnosis test kit, comprise the allergen protein chip detection technology that is used to this as pollen humuli scandentis disease specific diagnosis;

3) be used for the analysis of three-dimensional structure and the mensuration of antigenic determinant;

4) help having the acquisition of the main allergic protein of reorganization of identical sensitization or low irritability.

Description of drawings

Fig. 1 is the immunoblotting results of 28 routine pollen humuli scandentis positive serums to the pollen humuli scandentis crude extract, wherein 1～28 is 28 routine patients serums immunoblotting bands separately, and a is the pooled serum that derives from 28 routine positive serums, and b is the contrast of healthy volunteer's serum, c is a blank, M is the molecular weight of albumen standard, is respectively 97.0,66.0 from top to bottom, 45.0,30.0,20.1,14.4kDa;

Fig. 2 is the SDS-PAGE figure that target is collected component in the gel molecular exclusion chromatography, and M is a protein molecular weight standard among the figure, and 1 is the pollen humuli scandentis crude extract, and 2 are the pollen humuli scandentis crude extract through saltouing after handling, and 3 gel molecular exclusion chromatography target components are collected liquid;

Fig. 3 is that the first step and second of using HiTrap Q FF separation and purification to obtain goes on foot the SDS-PAGE figure that collects liquid, and M is a protein molecular weight standard among the figure, and 1～3 is the first step collection liquid, and 4 is second step collection liquid;

Fig. 4 is isoelectric focusing electrophoresis figure, and S-100 is that gel molecular exclusion chromatography target is collected component (repeating twice), and A is main allergic protein;

Fig. 5 is the typical curve of iso-electric point Mr;

Fig. 6 is the cross reaction detected result.

Embodiment

Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.

Determining of embodiment 1 main allergic protein of humulus pollen

1, the collection of pollen humuli scandentis disease patients serum and healthy volunteer's serum: the collection to pollen humuli scandentis disease patients serum and healthy volunteer's serum all has strict inclusion criteria.This research collects 28 routine pollen humuli scandentis disease patients serums altogether, and patient's inclusion criteria is as follows: 1) typical summer and autumn the pollen hypersensitivity medical history: seasonal rhinitis, eye conjunctivitis and (or) bronchial asthma, summer and autumn (7～October) morbidity; 2) pollen humuli scandentis immersion liquid intradermal test 〉=++ Pharmacia CAP system RAST FEIA detects pollen humuli scandentis sIgE 〉=II level (0.70kU/l) simultaneously.This research simultaneously collects 10 routine healthy volunteers' serum altogether, inclusion criteria is: no anaphylactic disease history (comprising medicine, food anaphylaxis and allergic rhinitis, bronchial asthma and other anaphylactic disease), Artemisia sieversiana Willd., humulus grass, PP (summer and autumn pollen mixture, containing oak-leaved goosefeet, hemp, Sunflower Receptacle and Siberian cocklebur pollen) intradermal test is all negative, routine blood test eosinophilic granulocyte and Total IgE in Serum are normal, the male sex's 6 examples, women's 4 examples, 22～45 years old age, year mean age (32 ± 4).All external immunological experiments that relate to human serum all adopt above-described pollen humuli scandentis disease patients serum and healthy volunteer's serum in this research.

2, saltout crude extract preparation of pollen humuli scandentis albumen: with 0.01MPBS damping fluid (pH8.0) with 1: 20 (mass/volume, w/v) ratio is extracted the pollen humuli scandentis of acetone degreaser drying in 4 ℃ of 24hr, when crude extract reaches at least 1mg/ml through concentrating with 80% saturation ratio ammonium sulfate precipitated protein, after sedimentary albumen is redissolved in 0.01MPBS damping fluid (pH8.0), through 10000g * 10min centrifuging and taking supernatant liquor, obtain the pollen humuli scandentis albumen crude extract of saltouing.

3, the determining of main allergic protein (employing western blotting method): the crude extract of will the above-mentioned pollen humuli scandentis albumen that makes saltouing prepares the glue separation through SDS-PAGE, and method is carried out with reference to the Laemmli method.Protein sample and sample buffer (2 *) are pressed 1: 1 mixed, and 100 ℃ are boiled 5min, sample on back 10000g * 5min centrifuging and taking supernatant.Use 15% separation gel, 5% concentrates glue (going up prescription according to " molecular cloning ") separates (DYCZ-23A type vertical electrophoresis groove, Beijing Liuyi Instrument Factory) to sample.Starting voltage 80V treats after the bromjophenol blue forward position enters separation gel voltage to be risen to 150V, and the constant voltage electrophoresis arrives the gel lower boundary until the bromjophenol blue forward position, stops electrophoresis.Take off gel and carry out half-dried transfer printing.Application DYCP-40C type transfer groove (Beijing Liuyi Instrument Factory) Towbin buffer (25mMTris, the 192mM glycine, 20% methyl alcohol, pH8.3), constant current transfer printing 90min (constant current value=glue area (cm ²) * 1.2mA), with albumen be transferred to pvdf membrane (aperture 0.45 μ m, MILLIPORE, U.S.A).Pvdf membrane is put into the 4 ℃ of sealings of spending the night of plate that fill confining liquid (5%BSA/PBS-T).With the pvdf membrane after the sealing be cut into 3mm wide little respectively with different patients serums' reactions (first antibody combine with antigen), incubation serum dilutes with the PBS-T work at 1: 3, room temperature is put the shaking table incubation that spends the night.One temperature resistance is educated and is finished the back with washings (PBS-T) washing 3 * 5min, after carry out incubation with horseradish peroxidase mark mouse anti human IgE antibody (two is anti-), room temperature 3hr, two anti-extent of dilution 1: 1000 (PBS-T makes diluent).Film two temperature resistances wash 3 * 5min with PBS-T after educating and finishing, and carry out the colour developing of DAB substrate, treat that it is available distilled water rinsing that protein band develops the color clear, termination reaction.The immunoblotting result as shown in Figure 1.1～28 is 28 routine patients serums immunoblotting bands separately among the figure, and a is the pooled serum that derives from 28 routine positive serums, and b is the contrast of healthy volunteer's serum, and c is a blank, and M is the molecular weight of albumen standard.The result can see that 12kDa position albumen is main allergic protein from the graph, and the positive reaction rate reaches 89%.

The separation and purification of embodiment 2 main allergic proteins

1, gel molecular exclusion chromatography

Chromatographic system: AKT Purifier10 (Amersham company)

Chromatography column: Sephacryl 100 prepacked columns (Amersham company)

Column volume 120ml

Length * diameter: 60cm * 16mm

Separating ranges: 10-100kDa

1) specimen preparation:

A, with the acetone degreasing of the pollen humuli scandentis of fresh collection, drying.

Extract (1/20w/v) 24hr with 0.01M PBS (pH8.0) magnetic force mild stirring under b, 4 ℃ of conditions.

C, 10000g * 30min carry out, and centrifugal (4 ℃) obtain supernatant liquor.

D, supernatant concentration to protein concentration carried out at least the ammonium sulfate precipitation precipitation of 80% saturation ratio during 1mg/ml, 5000g * 30min centrifugal treating, after the supernatant discarded protein precipitation is redissolved in 0.01M PBS (pH8.0) damping fluid, and the dialysis treatment of carrying out 2d (4 ℃) with this damping fluid to be to remove ammonium sulfate wherein, and the back concentrates protein concentration is reached about 12mg/ml.

2) balance liquid and elutriant: 0.05M PBS (pH7.2)

Elution requirement: V applied sample amount=1ml, flow velocity 0.5ml/min

Target components is that average retention volume is the elution peak of 87.25 ± 0.65ml, and this component has comprised the albumen of 10～38kDa scope.This separating step has good repeatability.The protein electrophoresis collection of illustrative plates as shown in Figure 2.

2, anion-exchange chromatography

Chromatographic system: AKT Purifier10 (Amersham company)

Chromatography column: HiTrap Q FF (Amersham company) (CV=1ml)

Buffering system:

0.02M BisTris-HCl (pH7.0) (column balance buffering liquid A, starting buffer)

0.02M BisTris-HCl (pH7.0)+2M NaCl (peak concentration elution buffer

B，elution?buffer)

Sepn process: the gel molecular exclusion chromatography is separated the target components collection liquid that obtains after distill water dialysis and concentration, reach 7.0 with the pH value of adjusting this protein solution with 5～10 times of dilutions of A damping fluid do, after 10000g * 10min is centrifugal, supernatant liquor is joined with among the good anion-exchange column HiTrap Q FF of A damping fluid balance, carry out linear gradient elution 15ml with the A damping fluid that contains 0～0.1M NaCl concentration gradient, with 0.1MNaCl constant concentration wash-out 15ml, then carry out the linear gradient elution of 20ml again with the A damping fluid that contains 0.1～0.2M NaCl concentration gradient.Wherein flow velocity is set at 1ml/min.

Separating resulting: successively gone out three peaks in linear gradient elution stage of 0～0.1MNaCl concentration range (elution volume 15ml) and 0.1MNaCl constant density wash-out stage (elution volume 15ml) subsequently, these three peaks all do not reach baseline separation, 1 is main peak, and 2,3 is two little acromions of successive.Linear gradient elution stage of 0.1～0.2MNaCl concentration range (elution volume 20ml) gone out one not sharp-pointed unimodal, be marked as 4.Wherein 4 is the elution peak of main allergic protein.1,2,3,4 electrophoretograms of respectively collecting component as shown in Figure 3.Obtaining this proteic molecular weight by ImageQuant TL v2003.03 software analysis is 12kDa.This component is made elisa assay, find that it is consistent with the proteic immunological experiment result of 12kDa among the embodiment 1.Illustrate to separate to obtain to be really main allergic protein of humulus pollen, this proteic N terminal amino acid sequence is NH ₄ ⁺-MDNPFENGMKA-COO ^-, do not find same protein by retrieval, so this albumen is a new albumen.

(isoelectric focusing electrophoresis is analyzed this proteic iso-electric point as shown in Figure 4), obtains typical curve (as shown in Figure 5): Y=3.5578x-11.736R by iso-electric point Mr by IEF ²=0.9971

The pI value of table 1 iso-electric point Mr and corresponding migration distance record

pI

?3.75

?4.55

?5.2

?5.85

?6.55

?6.85

?7.35

?8.15

?8.45

?8.65

?9.3

d(cm)

?1.5

?4.7

?6.4

?9

?11.5

?13.3

14.7

?16.7

?18

?19.2

?21.5

Iso-electric point according to typical curve derivation purifying allergic protein:

d＝5cm，pI＝4.7

The monoclonal antibody preparation of embodiment 3 main allergic protein of humulus pollen

Concrete operations are with reference to chapter 11 in Ahmedabad year chief editor " Immunization Update learns a skill and uses ": hybridoma technology and Monoclonal Antibody.Operating process is summarized as follows:

1) immune animal is got the female Sexual health BALB/c mouse immunity simultaneously in 36 ages in week.For the first time immune every injected in mice 10～15 μ g antigens.Freund's complete adjuvant is mixed with antigen 1 00 μ l equal-volume, with the abundant mixing of micro-stirrer, abdominal injection.

2) carry out the immunity second time after 2～4 weeks, also claim booster immunization.The antigen amount of booster immunization reduces by half, and uses Freund's incomplete adjuvant instead, but volume injected and method are constant.Booster immunization concurrence 4 times reaches requirement until serum titer.Satisfactory immune mouse should be had a rest one month before cytogamy at least, and serum titer is descended to some extent.

3) immunity was impacted in fusion in preceding 3 days for the last time, with PBS150 μ l dissolving antigen 50 μ g, through mouse tail vein injection.

4) get blood examination from eye socket after immune effect detects immune 2～3 times and survey serum titer.Draw blood plasma 2～5 μ l with sample injector, be diluted to 100 times with PBS, 1000 times, 2000 times, 4000 times.Detection method adopts ELISA.

5) cytogamy is collected myeloma cell and bone-marrow-derived lymphocyte respectively, ready myeloma cell and splenocyte are moved in the 50ml centrifuge tube, add the substratum that some do not contain serum, centrifugal, draw and to be placed on the centre of the palm behind the supernatant and to shake and make cell mass become loose pasty state.Centrifuge tube is placed 37 ℃ of water-baths, draw the PEG solution of 1ml 50%, slowly add in the cell, the limit edged stirs, and 1min adds.Leave standstill 1min and draw the substratum that preprepared 10ml does not contain serum again, slowly add,, reduce toxic action with dilution PEG.The limit edged slowly stirs, and slow earlier back is fast, and preceding 2min adds 2ml, and back 2min adds remaining substratum.Centrifugal, inhale and remove supernatant.Cell mass is shaken diffusing, add a spot of fresh IMDM substratum.

6) fused cell is inoculated and selectivity is cultivated, merging successful cell can survive in the HAT selective medium midium or long term, all the other not hybrid cell all with death.

7) detection of hybridoma detects those hybridomas that can secrete purpose antibody by the method for ELISA and immunoblotting.Drawing cell liquid nutrient medium supernatant detects.

8) cloning of positive hybridoma cell is cultivated the male hybridoma is carried out unicellular separation and Culture, adopts the semisolid medium method.

9) amplification of hybridoma and frozen that positive hybridoma cell need after the cloning cultivation are in time frozen to prevent the chromosome elimination of these cells is morphed.The amplification cultivation common perfect medium that contains 10% foetal calf serum, frozen employing contains the common perfect medium of 10%DMSO.

10) evaluation of monoclonal anti volume property identifies that content comprises the type and the subclass of affinity costant, specificity (cross reactivity), immunoglobulin (Ig).

11) production of monoclonal antibody is seeded to mouse peritoneal with hybridoma and prepares ascites, uses ascites separation and purification IgG antibody again.

The monoclonal antibody specificity analyses: purpose be intended to detect antibody whether also with purpose antigen outside other antigens react, this is the most important index of monoclonal antibody.Measuring method adopts ELISA (solid-phase enzyme-linked immune mensuration) method, following three kinds of antigens are carried out parallel bag quilt: main allergic protein of humulus pollen Hum s1, the immersion liquid of Artemisia sieversiana Willd. pollen allergen, the immersion liquid of oak-leaved goosefeet pollen allergen, coating protein concentration is 5 μ g/ml, the every strain monoclonal antibody that obtains is reacted with three kinds of envelope antigens simultaneously, if certain strain monoclonal antibody and two or more envelope antigen respond, illustrate that this strain monoclonal antibody has cross reactivity (promptly except can with can also combine with some antigen in Artemisia sieversiana Willd. pollen and/or the oak-leaved goosefeet pollen main allergic protein of humulus pollen Hum s1 combines), the purpose of this analysis method is intended to filter out monoclonal antibody with high specific (promptly only with main allergic protein of humulus pollen Hum s1 bonded antibody), therefore abandon the monoclonal antibody with cross reactivity, experimental result such as Fig. 6 show.Every row three Kong Weiyi group arranged side by side among Fig. 6, wrap s1 from left to right respectively by main allergic protein of humulus pollen Hum, the immersion liquid of Artemisia sieversiana Willd. pollen allergen, the immersion liquid of oak-leaved goosefeet pollen allergen, represent not homophyletic monoclonal antibody while and three kinds of antigen-reactives that wrap quilt for every group, have this strain monoclonal antibody that shows of two or more hole colour generations that cross reaction is arranged in three holes side by side, abandon, finally obtain 8 strains only with the antibody of main allergic protein of humulus pollen Hum s1 reaction.

Simultaneously, methods analyst by immunoblotting 8 strain antibodies that go out by the aforesaid method examination whether in all antigens that the pollen humuli scandentis allergenic extract is comprised, only combine with main allergic protein Hum s1, method is as follows: 1. the SDS-PAGE that runs the pollen humuli scandentis allergenic extract prepares electrophoresis; 2. half-dried being transferred on the pvdf membrane and with 5%BSA-PBST of albumen sealed; 3. carry out an anti-reaction with each strain monoclonal antibody after film being cut into wide little of 3mm; 4. an anti-reaction end back is washed film again and is carried out two anti-reactions with horseradish peroxidase mark sheep anti-mouse igg polyclonal antibody, carries out DAB at last and develops the color.The concrete operations of method can be with reference to the middle western blotting method that adopts of determining of main allergic protein of humulus pollen.The result shows that this 8 strain monoclonal antibody only combines with Hum s1 in the pollen humuli scandentis.

The mensuration of affinity costant: what affinity costant (Ka) reflected is antibody and antigen bonded ability.Under given conditions, for specific antigen-antibody system, Ka is an inherent, it is fast more that the big more reaction of this value reaches balance, and the relative concentration of immune complex is big more when reaching balance, and it is also few more to produce the required antigen amount of immune complex, and reaction was fast when promptly the antibody that avidity is high was used for Clinical Laboratory, consumption is few, and is highly sensitive.Mensuration to main allergic protein of humulus pollen Hum s1 monoclonal antibody affinity costant adopts dilution antibody act guestimate affinity costant, and concrete operations are as follows:

1. main allergic protein of humulus pollen Hum s1 is wrapped quilt, bag is 5 μ g/ml by concentration;

2. each strain antibody that will purify is done serial dilution;

3. each strain antibody is all got the antibody 100 μ l and the envelope antigen reaction of different concns, and along with the decline of antibody amount, combination rate constantly descends, and each strain antibody all can record one group of absorption value;

4. be that the ordinate zou antibody concentration is X-coordinate mapping with the reacting hole absorption value, from figure, find out combination rate from the vertex 50% o'clock corresponding antibody concentration that descends.The inverse of this concentration is the antibody affinity costant.

Obtain the affinity costant of 8 strain monoclonal antibodies thus, concrete numerical value sees Table, and wherein has the order of magnitude of two strain antibody affinity costants to reach 10 ⁹, the order of magnitude of four strain antibody affinity costants reaches 10 ⁸, the order of magnitude of two strain antibody affinity costants is 10 in addition ⁷

The immunoglobulin (Ig) of monoclonal antibody (Ig) type and subgroup identification: identify that monoclonal antibody Ig type and subclass adopt immune double diffusion method, concrete operations are as follows:

1. prepare 1% agarose solution with phosphate solution, pour into while hot in the little plate of diameter 35mm, the back punching of condensing, middle one, 6 on every side.

2. get hybridoma culture supernatant 1ml and add ammonium sulfate 0.3g precipitating antibody, the centrifugal 5min of 10000r/min inhales and removes supernatant, adds 50 μ l phosphate solutions, the dissolution precipitation thing.

3. get 10～15 μ l and add in the medium pore, peripheral hole adds antibody 10～15 μ l of anti-different I g class or subclass respectively, preserves moisture for 4 ℃ and places more than the 24hr, and the positive reaction of person that the precipitation line occurs shows that this antibody belongs to corresponding Ig class or subclass.

The result shows that the 8 strain monoclonal antibodies that filter out are IgG, and the subclass that 5 strain antibodies are wherein arranged is IgG1.

Above-mentioned experimental technique is all with reference to chapter 11 among the Ahmedabad year chief editor " Immunization Update learns a skill and uses ": hybridoma technology and Monoclonal Antibody are carried out.8 strain ideal hybridomas have been obtained altogether by aforesaid method, as shown in table 2, the monoclonal antibody that these hybridoma secretions produce all can be used for making up immunity detection reagent, thereby be used for detection of drugs and biotechnological formulation main allergic protein of humulus pollen content, determine drug dose and concentration.

The type of table 28 strain antibody or subclass and relative affinity constant

The cell strain numbering	Ig type and subclass	Relative affinity
			Hs?1 Hs?2 Hs?3 Hs?4 Hs?5 Hs?6 Hs?7 Hs?8	G1 G1 G1 G G G G1 G1	1.6×10 ⁷Lmol ^-1 1.6×10 ⁷Lmol ^-1 1.0×10 ⁸Lmol ^-1 2.0×10 ⁸Lmol ^-1 1.5×10 ⁹Lmol ^-1 1.4×10 ⁹Lmol ^-1 1.6×10 ⁸Lmol ^-1 3.2×10 ⁸Lmol ^-1

Reference

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Claims

1. the method for the main allergic protein matter of separation and purification from pollen humuli scandentis comprises step:

1) with after the pollen humuli scandentis degreasing, use the PBS buffer extraction, centrifugal supernatant, through saltout, redissolution, desalination, concentrate the pollen humuli scandentis protein crude extract;

2) adopt Sephacryl 100 chromatography columns of Amersham to carry out the gel molecular exclusion chromatography, wherein use 0.05M PBS upper prop and the wash-out of pH7.2, collecting retention volume is the elution peak of 87.25 ± 0.65ml;

3) reinforcing yin essence ion exchange chromatography, the chromatography column of wherein said reinforcing yin essence ion exchange chromatography are the HiTrap Q Sepharose FF of Amersham;

Wherein, the collection liquid of gel molecular exclusion chromatography is joined after pre-treatment in the described reinforcing yin essence ion exchange column after the balance, carry out linear gradient with the 0.02M BisTris-HCl of the pH7.0 that contains 0～0.1M NaCl concentration gradient and wash post, again with the constant post of washing of 0.02M BisTris-HCl of the pH7.0 of 0.1M NaCl, use the 0.02M BisTris-HCl wash-out of the pH7.0 of 0.1～0.2M NaCl at last, collect elution peak.