CN100535009C - Coli somatic polyclonal antibody, and its preparing method and use - Google Patents

Abstract

The invention discloses Escherichia coli thalli polyclonal antibody and its preparation method and application. The preparation method includes the following steps: separating out the Escherichia coli; putting it into physiological saline with asepsis glass bead and/or steel ball, waving, hatching at 80-120 degree centigrade for 1.5-3h, centrifuge, upper heat removing, sterilizing after PBS to gain Escherichia coli antigen; using it to immune animal; separating and purify the antiserum of the animal to gain the Escherichia coli thalli polyclonal antibody which has the advantages of high specificity, purity, titer( over 6400), saving for long, high precision, sensibility and less detecting steps for Escherichia coli by immunoassay in environment and food detecting field, wide application prospect.

Description

Coli somatic polyclonal antibody and preparation method thereof and application

Technical field

The present invention relates to antibody and preparation method thereof and application, particularly relate to a kind of coli somatic polyclonal antibody in the environment measuring field and preparation method thereof and its application in detecting intestinal bacteria.

Background technology

Intestinal bacteria are a kind of important indicator microoraganisms in environmental water quality monitoring and the food safety.At present, countries in the world and international organization all with coliform group count as important engine hygiene index, and strict restriction has been proposed its concentration in water.Regulation in World Health Organization's " water quality standard for drinking water " (second edition), intestinal bacteria in all tap water or heat-resisting colibacillus must not detect in the 100mL water sample arbitrarily; The existing U.S. country-level drinking water standard of water quality standard for drinking water stipulates that total intestinal bacteria (comprising excrement type and Emhorn colibacillus) are 0mg/L; Regulation total coli group and excrement colibacillus group must not detect in the 100mL water sample arbitrarily among State Standard of the People's Republic of China's " Drinking Water hygienic quality standard ".

At present, the coliform standard detecting method of generally acknowledging in the world is based on multitube fermentation method and filter membrane method.The detection method of the coliform group count that China stipulates in water and inspection for food hygiene standard also is multitube fermentation method and filter membrane method.But these two kinds of detection methods all exist sense cycle long, the shortcoming of complex operation, thereby be difficult to realize quick diagnosis to source of pollution.In recent years, number of research projects has been carried out at colibacillary rapid detection in water surrounding and the food in countries in the world, and wherein the ELISA detection method based on the immunological technique principle has presented good prospects for application.

Immunoassay technology is the specific reaction that utilizes between antigen and antibody, by the tracer of certification mark on reactant, and the Fast Detection Technique that antigen or antibody are qualitatively or quantitatively determined.According to the difference of mark substance, the immunologic detection method that is used for the testing environment pollutent at present mainly contains enzyme immunoassay technology, fluorescence immunoassay technology, chemiluminescence immunoassay technology, immuno-gold labeling technology, immunomagnetic bead technique etc.Wherein, enzyme immunoassay technology (Enzyme immunoassay, EIA) application in environmental area is comparatively extensive, and (Enzyme-linked immunosorbent assay ELISA) is the most frequently used measuring method again to the enzyme linked immunosorbent assay analysis method in such technology.This method has simple to operate, high specificity, and the sensitive advantage has advantage in colibacillary rapid detection rapidly.

Utilization polyclonal antibody ELISA methods such as Vandekerchove have detected pathogenic colon bacillus (EPEC), on single detection level (individual level), susceptibility and specificity that this method test obtains are respectively 80.0% and 98.4%, the susceptibility of (rabbit flock level) and specificity then are reduced to 79.2% and 85.2% (VANDEKERCHOVE DGF on a plurality of detection levels, KERR PG, CALLEBAUT AP, et.al.Development of a capture ELISA for the detection of antibodies toenteropathogenic Escherichia coli (EPEC) in rabbit flocks usingintimin-specific monoclonal antibodies[J] .Veterinary Microbiology, 2002.12,88 (4): 351-366.).Padhye adopts monoclonal antibody ELISA method to detect Escherichia coli O 157: H7 (PADHYE NV, DOYLE MP.Production and characterization of a monoclonalantibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7and O26:11[J] .J Clin Microbiol, 1991,29:99-103).Employing polyclonal antibody ELISA test kits such as Park have detected the Escherichia coli O 157 in the ight soil, compare with traditional Mai Kangkai medium therapy, the sensitivity of this test kit and specificity are respectively 91.2% and 99.5%, and the sensitivity of traditional detection method and specificity only are 82.4% and 100% (PARK CH, VANDEL NM, HIXON DL.Rapid immunoassay fordetection of Escherichia coli O157 directly from stool specimens[J] .J ClinMicrobiol, 1996,34:988-90).Ramadan etc. utilized the ELISA detection method to detect Escherichia coli O 157 (10-10 in 2 hours ⁸CFU/mL) (ABUKNESHA RA, DARWISH F.Coupling of enzymaticand immunoassay steps to detect E.coli:a new, highly sensitive tandemtechnique for the analysis of low levels of bacteria[J] .Talanta, 2005.1,65 (2): 343-348).But China Yao Fei etc. utilizes the indirect enzyme-linked immunosorbent adsorption method to detect the Escherichia coli O 157 of the non-cultivation conditions of living: H7, minimum detectable concentration is 10 ⁵CFU/mL (YAO Fei (Yao Fei), SHA Sha (Sha Sha), CHEN Gang (Chen Gang), et al.Indirect enzyme linked immunosorbent assay fordiagnosis of viable but nonculturable Escherichia coli O157:H7[J] .Journalof Ocean University of Qingdao (Qingdao Marine University's journal), 2001,31 (2): 211-214.).Application enzyme-linked immunosorbent assays such as Sun Kejiang have detected Escherichia coli O 157: H7, compare with the methods such as cultivation of routine, recall rate and susceptibility (the SUN Kejiang (Sun Kejiang) that all is largely increased, GUO Hong (Guo Hong), ZHANGDongfa (Zhang Dongfa), et al.Enzyme linked immunosorbent assay for detectingO157:H7[J] .Disease Surveillance (disease surveillance), 2001,16 (9): 353-355).Though the ELISA detection method is simple to operate, highly sensitive, but then need wide spectrum antibody (Broad spectrum antibody) as if the intestinal bacteria that detect all serotypes with this method, in addition, the mentioned reagent box wants to realize that commercialization still needs its reliability is done further check, and present examination criteria disunity still, therefore, commercialization is subjected to very big restriction.

FDA has been approved the immunity detection reagent of the hemorrhagic colibacillus O157:H7 of commodity in useization.China only China Veterinary Drugs Supervisory Inst. has developed the ELISA detection kit at colon bacillus K88, K99 and 987P antigen respectively, be used to control cub colibacillosis (China Institute of Veterinary DrugControl (not having concrete author) .Research and application of enzyme linkedimmunosorbent assay for detecting E.coli kit.Bulletin of Agricultural Scienceand Technology (agricultural science and technology communication), 1997, the 8 no volume phase page numbers).By in above many articles as seen, at present domestic and international research and use spininess to pathogenic Escherichia coli O 157: H7 does not also have the ELISA detection kit at total intestinal bacteria exploitation, and requires total intestinal bacteria that are of detection in the environment measuring standard.Simultaneously, each result of study shows, the detectability that is directed to each technology that intestinal bacteria develop all is difficult to satisfy the requirement of environment measuring.Therefore, press for and be used to detect total colibacillary ELISA test kit in a kind of environment measuring field.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of simple and easy to do coli somatic polyclonal antibody.

For achieving the above object, the present invention takes following design: a kind of preparation method of coli somatic polyclonal antibody may further comprise the steps:

1) isolates intestinal bacteria, concrete grammar is: sample is inoculated on the beef extract-peptone nutrient agar earlier and cultivated 12-36 hour down at 36-38 ℃, to coat on the fuchsin sulfite substratum at the bacterium colony that the beef extract-peptone solid medium grows again and cultivate 24-48 hour down at 36-38 ℃, then single bacterium colony of the purplish red colour band metalluster that on the fuchsin sulfite substratum, grows of picking, be inoculated in once more on the fuchsin sulfite substratum and cultivated 24-48 hour down at 36-38 ℃, single bacterium colony of the purplish red colour band metalluster of picking then, be inoculated in to have added in the lactose protein peptone substratum that mass percentage concentration is the 1.4-1.8% purpurum bromocresolis and cultivated 12-36 hour down at 36-38 ℃, treat that the purple in the lactose protein peptone substratum takes off, bacterium liquid is inoculated on the beef extract-peptone nutrient agar inclined-plane, the bacterium colony that grows is carried out gramstaining, observe ne ar, to be accredited as colibacillary bacterium colony and be inoculated on the methylene blue substratum of Yihong at 36-38 ℃ and cultivated 12-36 hour down, bacterium colony darkly purple be intestinal bacteria;

2) the intestinal bacteria cultivation is placed in the physiological saline that contains sterile glass beads and/or steel ball, shakes, under 80-120 ℃, hatched 1.5-3 hour then, centrifugal, abandon supernatant, washing, centrifugal again, abandon supernatant, bacterial sediment is sterilized to it with the resuspended back of PBS, obtain coli somatic antigen;

3) with the coli somatic antigen-immunized animal;

4) from separation, purifying antiserum(antisera) through the animal of immunity, obtain coli somatic polyclonal antibody.

There are multiple intestinal bacteria in the physical environment, particularly sanitary sewage, thereby in above-mentioned preparation method's step 1), at first need to isolate the multiple intestinal bacteria that are present in the testing sample, so that the polyclonal antibody of preparation has broad spectrum.

Step 2) selection that is used to cultivate colibacillary substratum in is diversified, as beef extract-peptone nutrient agar, LB substratum etc., is preferably the beef extract-peptone nutrient agar, and culture condition can be at 36-38 ℃ and cultivated 12-36 hour down; The size of granulated glass sphere and steel ball and quantity are decided by container size and amount of liquid, have no special requirements; Incubation temperature is preferably 100 ℃, and incubation time is preferably 2-2.5 hour; Centrifugal condition all is preferably centrifugal 8-12min under 4000-6000rpm; Wash with stroke-physiological saline solution; The condition that O antigen is sterilized can be heating 15-25min under 120 ℃.

The selection of immunization method is diversified in the step 3), and as the subcutaneous multi-point injection method in back, intraperitoneal injection etc., immunizing dose can be 0.3-2.0mL/, and only (concentration is 10 ⁸-10 ¹⁰Cfu/mL), in addition, the immune animal that is used to prepare coli somatic polyclonal antibody can be immune animal commonly used such as rabbit, chicken, mouse, sheep or horse.

For obtaining quite good detecting effectiveness, also should measure the sero-fast antibody titer of immune animal in the step 4) every 7-10 days, when antibody titer reaches maximum value, can be from separation, purifying antiserum(antisera) through the animal of immunity; The method that described antagonistic Serum carries out purifying can be methods such as saturated ammonium sulphate salt precipitation method and affinity chromatography.

Coli somatic polyclonal antibody with method for preparing is that the present invention needs protection.

Coli somatic polyclonal antibody of the present invention can be used in the colibacillary immunodetection.

Described colibacillary immunological detection method can adopt enzyme-linked immunosorbent assay (ELISA), immuno-gold labeling technology, chemiluminescent immunoassay technology or fluorescence immunoassay technology etc.

Those skilled in the art know, and for solid state reaction, polyclonal antibody of the present invention can be fixed in solid phase carrier, also testing sample can be fixed on the solid phase carrier.Reaction is at room temperature carried out usually, needs to wash not the step with polyclonal antibody bonded sample of the present invention in the testing process.

For liquid phase reaction, can in the testing sample in being in specific buffer system, directly add polyclonal antibody of the present invention usually, then (as room temperature) takes place under the interactional temperature react being suitable for antigen-antibody.

Those skilled in the art know how to select corresponding second antibody according to the source of polyclonal antibody.Described second antibody can be a labelled with radioisotope, includes but not limited to use be selected from: ³²P, ¹²⁵I, S, ²H etc.; Can be non-labelled with radioisotope also, include but not limited to use be selected from: marks such as horseradish peroxidase, alkaline phosphatase, vitamin H, Streptavidin.The detection reagent of using in the detection method of the present invention depends on the marker of the second antibody that testing process is used, and those skilled in the art know how to select suitable detection reagent.

Described colibacillary ELISA detection method can comprise the steps:

1) with the testing sample bag by elisa plate, wash plate;

2) sealing is washed plate through the elisa plate of bag quilt;

3) add the intestinal bacteria somatic polyclonal antibody, wash plate;

4) add ELIAS secondary antibody, wash plate;

5) add the substrate colour developing;

6) termination reaction;

7) measure OD ₄₅₀Value.

Reaction conditions in the above-mentioned detection method and reagent all can be selected according to ordinary method.

The anti-anti-rabbit anti-antibody that can be horseradish peroxidase or alkaline phosphate ester enzyme labelling of in the step 4) two.

The present invention also provides a kind of detection colibacillary ELISA test kit.

The colibacillary ELISA test kit of detection provided by the present invention can comprise above-mentioned coli somatic polyclonal antibody.

Described test kit also can comprise the ELIAS secondary antibody of described coli somatic polyclonal antibody.

Described ELIAS secondary antibody can be goat anti-rabbit igg etc.

The described mark two anti-enzymes that are used for can be marker enzymes such as horseradish peroxidase or alkaline phosphatase, are preferably horseradish peroxidase, and horseradish peroxidase can be crosslinked on antibody by glutaraldehyde method or periodic acid method.

Also can comprise the colour developing liquid of being made up of colour developing liquid A and colour developing liquid B in the test kit, described colour developing liquid A liquid is hydrogen peroxide or urea peroxide solution, and described colour developing liquid B liquid is O-Phenylene Diamine or 3 ', 3 ', 5 ', and 5 '-tetramethyl biphenyl amine aqueous solution.

Use for convenient, described test kit also can comprise the washings that detects usefulness, as conventional washing reagents such as PBST; Confining liquid is as 1%BSA etc.; Coating buffer is as 0.05M carbonate buffer solution (pH 9.6) etc.

The invention provides a kind of coli somatic polyclonal antibody and preparation method thereof.In preparation method of the present invention, intestinal bacteria separate from physical environment, particularly in the sanitary sewage, thereby are colibacillary representative flora types in the environment, but the intestinal bacteria based in this polyclonal antibody specific recognition environment that obtains have broad spectrum; In addition, this preparation method also has simply, advantage with low cost.Experimental results show that, coli somatic polyclonal antibody with the inventive method preparation has the specificity height, purity and the height of tiring (greater than 6400), advantage with long preservation period, use it in environment and the food inspection field in the colibacillary immunodetection, to have advantages such as accuracy, sensitivity height, have a extensive future.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

Fig. 1 is the SDS-PAGE detected result of coli somatic polyclonal antibody

Fig. 2 is the titration result of coli somatic polyclonal antibody

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.All with the distilled water preparation, it is standby that packing packages the back sterilization for all substratum, and sterilising conditions is at 115 ℃ of 20-30min that sterilize down.

The preparation of embodiment 1, coli somatic polyclonal antibody and titration thereof

One, preparation coli somatic polyclonal antibody

The method of current series invention prepares coli somatic polyclonal antibody, may further comprise the steps:

1, from sanitary sewage, isolates intestinal bacteria

1) gathers water sample: gather water sample from 4 sanitary sewage disposal factories of Beijing (water inlet of Qinghe sewage work, Xiao Jia river sewage work, northern river sewage work and Tsing-Hua University be the sanitary sewage water sample in the school) respectively, be total to water sampling 16 points, every factory 4 points.The plastic sample bottle use in sampling, reduces during water intaking and air duration of contact as far as possible, grasps the sample bottle bottom, and bottle is immersed the underwater rapidly, then bottleneck is turned to water (flow) direction, treats that water sample was filled to bottle at long-pending 2/3 o'clock, bottle cap beyond the Great Wall in water, the taking-up water surface.The water sample of fetching is stored in 4 ℃ of refrigerators, and the shelf time should be above 6 hours.

2) colibacillary separation and evaluation

Adopt the selectivity cultural method to isolate intestinal bacteria from the water sample of step 1) collection, concrete grammar may further comprise the steps:

A. the water sample streak inoculation is cultivated 24 hours (rejuvenation) for last 37 ℃ in beef extract-peptone nutrient agar (NaCl2.5g, agar 10g, water 500mL, pH 7.2 for extractum carnis 2.5g, peptone 5.0g);

B. will coat fuchsin sulfite substratum (peptone 10g at the bacterium colony that the beef extract-peptone solid medium grows, yeast extract 5g, extractum carnis 5g, lactose 10g, agar 20g, dipotassium hydrogen phosphate 3.5g, sodium sulphite anhydrous 99.3 5g, 5% magenta ethanolic soln 20mL, water 1000mL, pH to 7.2-7.4) go up under 37 ℃, to cultivate and separated in 24-48 hour;

C. the purplish red colour band metalluster list bacterium colony that on the fuchsin sulfite substratum, grows of picking, streak inoculation is cultivated down at 37 ℃ on the fuchsin sulfite substratum and was separated in 36 hours again;

D. the isolated purplish red colour band metalluster list bacterium colony of picking step C, be inoculated in lactose protein peptone substratum (the peptone 5g that has added purpurum bromocresolis, extractum carnis 1.5g, lactose 2.5g, sodium-chlor 2.5g, 0.5mL 1.6% purpurum bromocresolis ethanolic soln, water 500mL, pH 7.2) in cultivated about 24 hours down at 37 ℃;

E. treat that the purple in the lactose protein peptone substratum takes off among the step D, the bacterium liquid in the picking substratum is seeded in it on beef extract-peptone nutrient agar inclined-plane;

F. the bacterium colony that grows is carried out gramstaining on beef extract-peptone nutrient agar inclined-plane, under opticmicroscope, observe ne ar, will be accredited as colibacillary bacterium colony and be coated with again and be inoculated in Yihong methylene blue substratum (semi-lactosi 5g, Tryptones 2.5g, NaCl 2.5g, K ₂HPO ₄1g, Eosin Y 0.2g, methylene blue 0.05g, agar 7.5g, water 500mL, pH 7.2) on, to cultivate about 24h down at 37 ℃ and observe the color form, bacterium colony is purple darkly, and glossy or lacklustre is intestinal bacteria, cryopreservation, standby (generally can store about 6 months).The intestinal bacteria that obtain with aforesaid method have comprised representational intestinal bacteria in the water surrounding, can be in order to characterize the total intestinal bacteria in the water surrounding.

2, the antigenic preparation of coli somatic

It is 10 that the isolating intestinal bacteria of step 1 are made concentration ⁸-10 ¹⁰The bacterial suspension inoculation of cfu/mL is in beef extract-peptone nutrient agar (extractum carnis 2.5g, peptone 5.0g, NaCl 2.5g, agar 10g, water 500mL, pH 7.2) on, cultivated 12-24 hour down at 37 ℃, from culture dish, scrape lawn, place the sterile glass beads that contains and the 150mL angle flask of 20mL physiological saline, fully shake and the mixing thalline, after hatching 2-2.5 hour under 100 ℃, bacterium liquid is moved in 2 10mL centrifuge tubes then, the centrifugal 10min of 5000rpm, abandon supernatant, again with stroke-physiological saline solution washing, the centrifugal 10min of 5000rpm then, abandon supernatant, bacterial sediment is resuspended with PBS, bacterium liquid is poured in the flask of sterilization 120 ℃ down heating 20min sterilize, obtain coli somatic antigen.

3, immune animal

The coli somatic antigen that step 2 is obtained adopts 6 hypodermic injections in back (near oxter, back, each both sides of inguinal region of lymphoglandula) to carry out immunity to 10 female new zealand white rabbits of growing up, immunity is got rabbit auricular vein blood in preceding 1 week, separation of serum is as negative control (Neg), immunizing dose is 1.0mL/ time, filling 1.0mL freund adjuvant (available from sigma company) is as immunological adjuvant during each immunity, respectively at the 7th, 14,21 and 28 day booster immunization once (immunization protocol sees Table 1) again behind the initial immunity.

Table 1 immunization protocol

4, the acquisition of coli somatic polyclonal antibody

1) titration

In the immunologic process, regularly (after the initial stage immunity every 7 days, detect every day after treating immunity five times) from the ear edge vein exploitating blood of immune rabbit, blood sampling volume is 1mL/, separation of serum, (tire (titer) (claiming titre again) of antibody is an important indicator estimating the antibody performance, is a sxemiquantitative index that is usually used in expressing specific antibody relative content in the antiserum(antisera) with the antibody titer in the indirect elisa method detection serum.Tiring is meant under certain condition, and antiserum(antisera) is through a series of dilutions and quantitative antigen-reactive, when positive antiserum(antisera) light absorption value sero-fast extension rate during greater than the contrast light absorption value of 2.1 times of negative serums.) to investigate immune effect, concrete steps are as follows:

A. wrap quilt: with 0.05M pH 9.6 sodium bicarbonate buffer solution (Na ₂CO ₃0.159g, NaHCO ₃0.294g, ddH ₂O 100mL) with antigen coated 96 orifice plates of coli somatic, every hole package amount is 100 μ L, 37 ℃ of incubation 2h;

B. sealing: (include 0.01M pH7.4 phosphoric acid salt-NaCl damping fluid (PBS) of 0.05%Tween-20, filling a prescription is: NaCl 8.0g, KH with the PBST washings ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl 0.2g, Tween-20 0.5mL, water is settled to 1L) cleaning of enzyme target 3 times, each 3min, it is 1% bovine serum albumin (BSA) confining liquid 100 μ L that mass percentage concentration is added in every again hole, 37 ℃ of incubation 2h;

C. add antibody: with PBST washings cleaning of enzyme target 3 times, each 3min adds the antiserum(antisera) 100 μ L of above-mentioned separation from immune rabbit, 37 ℃ of incubation 1h again;

D. enzyme-added mark anti-antibody: with PBST washings cleaning of enzyme target 3 times, each 3min adds goat-anti rabbit anti-antibody (available from Sigma) the 100 μ L of horseradish peroxidase-labeled, 37 ℃ of incubation 1h again;

E. add substrate colour developing: with PBST washings cleaning of enzyme target 3 times, each 3min adds 3 ', 3 ' again, and 5 ', 5 '-tetramethyl benzidine (3,3 ', 5,5 '-tetramethylbenzidine, TMB) solution (Na ₂HPO ₄142mg, citric acid 105mg, TMB 2mg, 30%H ₂O ₂7.5 μ l, distilled water 5mL) 100 μ L, reaction 5min;

F. termination reaction and OD ₄₅₀PH-value determination pH: add H ₂SO ₄Stop buffer (H ₂SO ₄28mL, ddH ₂O 500mL) after the termination reaction, takes out and put into microplate reader, read the absorbancy under the 450nm.

2) separate antiserum(antisera)

When tiring of immune rabbit antibody reaches the highest, method with the carotid artery bloodletting is taken a blood sample in a large number and (is noted: before a large amount of blood samplings, earlier with rabbit fasting 24h in case blood fat is too high), get and at room temperature place about 2h behind the blood it is solidified, use the centrifugal 10min of supercentrifuge 11000rpm then, get the supernatant packing, in-20 ℃ of freezings (or in 4 ℃ of refrigerations).

3) sero-fast purifying

A. carry out purifying with saturated ammonium sulphate salt precipitation method

At first use saturated ammonium sulphate salt precipitation method to step 2) isolating antiserum(antisera) carries out preliminary purification, and concrete grammar may further comprise the steps:

(1) gets antiserum(antisera) sample 20mL, add equivalent PBS (NaCl 8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl 0.2g, water is settled to 1L, pH 5.0), in stirring gently with glass stick on ice, slowly, dropwise add SAS (saturated ammonium sulphate solution) 40mL of 4 ℃ of precoolings, in 4 ℃ of refrigerators, leave standstill 12-24h (annotate: after adding isopyknic SAS final concentration become original content 50%);

(2) 4 ℃, the centrifugal 10min of 12000rpm abandon supernatant, and throw out is dissolved with 12mL PBS, slowly, dropwise add the 8mL SAS of 4 ℃ of precoolings, and 4 ℃ leave standstill 1h;

(3) repeating step (2) is centrifugal, with 13.4mL PBS dissolution precipitation, drips 6.6mL SAS, and 4 ℃ leave standstill 1h;

(4) repeating step (2) is centrifugal, will precipitate the dissolving with PBS, the dialysis tubing of packing into;

(5) in 4 ℃ of refrigerators, dialyse with the PBS of 25 times of volumes, during every 3h change liquid once, change liquid altogether 3 times, until using BaCl ₂Detecting dialyzate does not have till the white precipitate.

B. carry out purifying with affinity chromatography

Use albumin A affinity column (HiTrap rProtein A FF) available from AmershamBiosciences company to do to the antiserum(antisera) that carries out preliminary purification with saturated ammonium sulphate salt precipitation method in the steps A and be further purified, concrete grammar may further comprise the steps:

(1) prepares collection tube, to waiting that collecting part adds 120 μ l 1M TrisHCl (pH 9.0)/mL;

(2) with initial damping fluid (the binding product of HiTrap rProtein A FF chromatography column) filled syringe, with the distilled water wash alcohol sanitas of at least 5 times of column volumes;

(3) wash post with the elution buffer (the binding product of HiTrap rProtein A FF chromatography column) of 5 times of column volumes and make column regeneration;

(4) with coupling buffer (the binding product of HiTrap rProtein A FF chromatography column) the balance pillar of 5-10 times of column volume;

(5) sample is crossed post, with syringe sample is injected end interface on the pillar;

(6) wash post (noting: avoid overwass) with the coupling buffer of 5-10 times of column volume;

(7) with the elution buffer wash-out of 2-5 times of column volume, obtain coli somatic polyclonal antibody.

Two, the detection of coli somatic polyclonal antibody

1, protein content determination

The protein content of the coli somatic polyclonal antibody that the use spectrophotometer prepares with the inventive method in the determination step one under 280nm, somatic antibody 2.1mg/mL shows to have obtained the higher coli somatic polyclonal antibody of protein content as a result.

2, SDS-PAGE electrophoresis detection

Coli somatic polyclonal antibody to the step 1 preparation carries out the SDS-PAGE electrophoresis detection again, specifically detects step and is: successively clean the glass plate with liquid detergent, tap water, distilled water, dehydrated alcohol, dry with thieving paper; Join glue: the Tris glycine SDS-PAGE separation gel solution of configuration 10mL 15% and the Tris glycine SDS-PAGE of 4mL 5% concentrate sol solution, sample preparation liquid (1g SDS, 5mL glycerine, 0.5mg BPB, 2.5mL mercaptoethanol, the TrisHCl damping fluid of 10mL pH6.8 adds water to 50mL); Behind the abundant mixing of separation gel solution, reposefully it is added 4.5mL from a side, on liquid level, inject one deck water then,, impel polymerization and eliminate the formation of gel meniscus, make gel surface smooth with secluding air; After in 37 ℃ of incubators, placing 40min water is outwelled, blotted, inject concentrated glue, carefully insert comb then immediately, place 30min in 37 ℃ of incubators to glass plate top with filter paper; Fill electrophoretic buffer toward the electrophoresis inside groove earlier when pulling out comb, make the comb bore deformation, remove the gel seal frame simultaneously, can go up sample behind the 30min in order to avoid there is bubble to enter the comb hole; During sample preparation, mix at 1: 1, it is boiled 10min together with standard protein Marker under 100 ℃, be cooled to room temperature, centrifugal (removing some insoluble materials in the sample) with 2 * sample preparation liquid and sample; During last sample, applied sample amount is 10 μ g/ holes; To concentrate glue and separation gel and under 80V and 160V, carry out electrophoresis respectively; When arriving the sheet glass bottom, the tetrabromophenol sulfonphthalein forward position stops electrophoresis; When getting glue, sled removes the glass plate, will concentrate glue and scrape off gently, avoids separation gel is scratched, and separation gel is unloaded, and upper left corner cut is used pure water rinsing; Dyeing and when decolouring will be outwelled staining fluid behind the separation gel heated by microwave 1-2min, again with microwave heating 3min * 2 (amplification quantity sponge in the plate) of decolouring, shaking table vibration cooling; After the gel decolouring is clean, destainer reclaims through gac, can scan behind the gel bubble clear water, (swimming lane 0 is a coli somatic polyclonal antibody to the result as shown in Figure 1, swimming lane M is the protein standard molecular weight), the heavy chain of gained coli somatic polyclonal antibody and light chain are clear as can be seen, and foreign protein content is few, show that the coli somatic polyclonal antibody with the inventive method preparation has higher purity.

3, titration

With with step 1 in identical indirect elisa method measure with the tiring of the coli somatic polyclonal antibody of the inventive method preparation, the antibody in step c becomes the coli somatic polyclonal antibody, all the other steps are all identical.The titration result is (the negative contrast of Neg as shown in Figure 2,0 is coli somatic polyclonal antibody), produced stronger immune response with coli somatic antigen immune New Zealand white rabbit, the coli somatic polyclonal antibody that obtains has higher absorption value to the coli somatic antigen of bag quilt, show that antibody can have stronger atopy with antigen, the coli somatic polyclonal antibody that is obtained is tired higher, greater than 6400.

The ELISA test kit that embodiment 2, usefulness contain coli somatic polyclonal antibody detects intestinal bacteria

One, contains the preparation of the ELISA test kit of coli somatic polyclonal antibody

With the coli somatic polyclonal antibody of embodiment 1 preparation and goat anti-rabbit igg as process the horseradish peroxidase-labeled of second antibody, A liquid: 30%H develops the color ₂O ₂1mL, colour developing B liquid: mass percentage concentration be 1% 3 ', 3 ', 5 ', 5 '-tetramethyl benzidine (TMB) liquid 1mL (is dissolved in dimethyl sulfoxide (DMSO) (Dimethylsulfoxide with TMB, DMSO) (or dimethyl formamide (Dimethyl formamide, DMF)) obtain), washing and diluent PBST 50mL, confining liquid 1%BSA 10mL, coating buffer 0.05M pH 9.6 sodium bicarbonate 50mL, H ₂SO ₄Stop buffer 10mL packs jointly, obtains detecting colibacillary ELISA test kit.

Two, colibacillary detection

With Tsing-Hua University in the school sanitary sewage be example, with the test kit of step 1 preparation the intestinal bacteria in the environment are measured, concrete grammar may further comprise the steps:

(1) bag is by elisa plate: with 0.05M pH 9.6 sodium bicarbonate buffer solutions the sewage water sample is coated in 96 orifice plates, and every hole 100 μ l, 37 ℃ of placements 2 hours use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;

(2) elisa plate of quilt has been wrapped in sealing: the elisa plate of BSA sealing bag quilt with 1%, and 100 μ l/ holes, 37 ℃ are incubated 30 minutes;

(3) add coli somatic polyclonal antibody (through 200 times of dilutions) the 100 μ l/ holes of implementing 1 preparation on elisa plate, 37 ℃ of insulations 1 hour use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then.

(4) add the goat anti-rabbit igg of horseradish peroxidase-labeled, 100 μ l/ holes, 37 ℃ of insulations 30 minutes use 10mM PBS-0.05%Tween 20 (pH7.4) to give a baby a bath on the third day after its birth time then;

(5) adding the horseradish peroxidase substrate solution (adds 0.2mL TMB solution, adds 20 μ l 30%H again in 9.8mL pH5.0 citric acid-phosphate buffered saline buffer ₂O ₂, shake up), 50 μ l/ holes, color development at room temperature 5 minutes;

(6) add 2N H ₂SO ₄50 μ l/ hole termination reactions;

(7) measure OD ₄₅₀Value.

OD as a result ₄₅₀Value is 0.6, the typical curve that contrast is drawn with the Escherichia coli bacteria liquid of concentration known, and the intestinal bacteria amount in the sample is about 10 ⁴/ mL, the test kit that shows coli somatic polyclonal antibody of the present invention and contain this antibody can be used in the colibacillary immunodetection.

Claims (4)

1, the antigenic method of a kind of preparation coli somatic may further comprise the steps:

1) separating Escherichia coli in the life sewage certainly: the sanitary sewage sample is inoculated on the beef extract-peptone nutrient agar cultivated 12-36 hour down earlier at 36-38 ℃, to coat on the fuchsin sulfite substratum at the bacterium colony that the beef extract-peptone nutrient agar grows again and cultivate 24-48 hour down at 36-38 ℃, then single bacterium colony of the purplish red colour band metalluster that on the fuchsin sulfite substratum, grows of picking, be inoculated in once more on the fuchsin sulfite substratum and cultivated 24-48 hour down at 36-38 ℃, single bacterium colony of the purplish red colour band metalluster of picking then, be inoculated in to have added in the lactose protein peptone substratum that mass percentage concentration is the 1.4-1.8% purpurum bromocresolis and cultivated 12-36 hour down at 36-38 ℃, treat that the purple in the lactose protein peptone substratum takes off, bacterium liquid is inoculated on the beef extract-peptone nutrient agar, the bacterium colony that grows is carried out gramstaining, observe ne ar, to be accredited as colibacillary bacterium colony and be inoculated on the methylene blue substratum of Yihong at 36-38 ℃ and cultivated 12-36 hour down, bacterium colony darkly purple be intestinal bacteria;

2) the intestinal bacteria cultivation is placed in the physiological saline that contains sterile glass beads and/or steel ball, shakes, under 80-120 ℃, hatched 1.5-3 hour then, centrifugal, abandon supernatant, washing, centrifugal again, abandon supernatant, bacterial sediment is sterilized to it with the resuspended back of PBS, obtain coli somatic antigen.

2, method according to claim 1 is characterized in that: described step 2), being used to cultivate colibacillary substratum is the beef extract-peptone nutrient agar, and culture condition is to cultivate 12-36 hour down at 36-38 ℃.

3, method according to claim 1 is characterized in that: described step 2), the condition that bacterial sediment is sterilized is heating 15-25min under 120 ℃.

4, method according to claim 1 is characterized in that: described step 2), incubation temperature is 100 ℃, and incubation time is 2-2.5 hour; Centrifugal condition is centrifugal 8-12min under 4000-6000rpm; Wash with stroke-physiological saline solution.

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