CN1811433A - Method for detecting gymnosperm pollen in-tube antigen - Google Patents
Method for detecting gymnosperm pollen in-tube antigen Download PDFInfo
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- CN1811433A CN1811433A CN 200610003166 CN200610003166A CN1811433A CN 1811433 A CN1811433 A CN 1811433A CN 200610003166 CN200610003166 CN 200610003166 CN 200610003166 A CN200610003166 A CN 200610003166A CN 1811433 A CN1811433 A CN 1811433A
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Abstract
The present invention discloses a method for detecting gymnosperm pollen tube interior antigen. Said method includes the following steps: (1), using phosphoric acid buffer solution containing paraformaldehyde to fix pollen tube, using zymolysis solution containing cellulose and macerozyme to zymolyze gymnosperm pollen tube, using Triton X-100 or NP-40 aqueous solution whose mass percentage concentration is 0.2-1% to wash the pollen tube; and (2), making the pollen tube treated by step 1 and the correspondent antibody for resisting the antigen to be tested undergo the process of immunofluorescence reaction, if the fluorescence is detected, the gymnosperm pollen tube interior contains antigen to be tested and can obtain the location condition of said antigen to be tested.
Description
Technical field
The present invention relates to a kind of method that detects gymnosperm pollen in-tube antigen, particularly a kind of method of utilizing immunofluorence technic to detect gymnosperm pollen in-tube antigen.
Background technology
Pollen tube carries and is transmitting spermatid and arrive ovule as the carrier of male germ unit in the Anthophyta fertilization process, realizes smart ovum combination, finishes amphigamy.Pollen tube is a kind of typical tool apical growth characteristic and self-existent haploid cell, presents the column polar growth pattern of homogeneous when growth in vitro, thereby is considered to the desirable cell system of biological technical field research polar growth.In addition, pollen tube is as a mode system, and for the growth of further investigation cell polarity, cell-cell interaction and signal conduction are also significant.Because the pollen tube cell membrane mainly is made up of polysaccharide such as cellulose, callose, pectic substance and some protein substances, the detection and location of born of the same parents' endoantigen are subjected to the restriction of cell wall substance, and allogenic material is difficult to enter in the born of the same parents to take place to send out with antigen and answers.In addition, for some proteantigens, owing in fixation procedure, be easy to cause the antigen inactivation, and the restriction of required time-temperature condition in the antigen-antibody reaction process, make the detection and location of pollen tube endoantigen difficult, and the efficient ways that does not also have relevant gymnosperm pollen in-tube antigen detection and location so far causes the relative angiosperm of the biological study relevant with gymnosperm relatively to lag behind.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects gymnosperm pollen in-tube antigen.
The method of detection gymnosperm pollen in-tube antigen provided by the invention may further comprise the steps:
1) with the phosphate buffer that contains paraformaldehyde with pollen tube fixing after, with the enzymolysis liquid enzymolysis gymnospermae pollen pipe of cellulase and macerozyme, be TritonX-100 or the NP-40 solution washing of 0.2%-1% again with mass percent concentration;
2) will carry out Immunofluorescence Reactions by the antibody corresponding with anti-determined antigen through the pollen tube that step 1) is handled, as detect fluorescence, then the gymnospermae pollen pipe contains this determined antigen and obtains the location situation of this determined antigen.
In the step 1), in order to obtain higher sensitivity and the signal to noise ratio (S/N ratio) of Geng Gao, in the described phosphate buffer that contains paraformaldehyde, the quality percentage composition of paraformaldehyde is 2-4%.
Described regular time can be 1-2 hour.
The mass percent concentration of the paraformaldehyde that described fixedly pollen tube is used is preferably 2%, and the set time is preferably 2 hours.
The enzymolysis liquid of described cellulase and macerozyme contains the cellulase that mass percent concentration is 1.5-3%, mass percent concentration is the macerozyme of 1.2%-2%, pH5-6,15mM 2-(N-morphine quinoline) ethyl sulfonic acid (MES), 400mM sweet mellow wine, 5mM lime chloride and 1 μ gmL
-1Protease inhibitors.
Described protease inhibitors can be aprotinin, pepstatin A, chymostatin or leupeptin.
The mass percent concentration of described cellulase is preferably 1.5%; The mass percent concentration of described macerozyme is preferably 1.2%.
The time of described enzymolysis gymnospermae pollen pipe can be 8-15 minute, is preferably 10 minutes.
The mass percent concentration of described TritonX-100 is preferably 0.2%.
Described cellulase can be commercially available various cellulases, preferred cellulase R-10; Described macerozyme can be commercially available various macerozymes, preferred macerozyme R-10.
Step 2) in, it is that the pollen tube of handling through step 1) is transferred on the microslide of poly-D-lysine bag quilt that the described pollen tube antibody of handling through step 1) corresponding with anti-determined antigen carries out Immunofluorescence Reactions, at room temperature sealed 1-2 hour with the 1-3% bovine serum albumin solution, after TritonX-100 through containing 0.2%-1% or the phosphate buffer of NP-40 fully wash, hatched 2-12 hour under 0-4 ℃ or the room temperature in anti-at one of anti-determined antigen, transfer in the two anti-solution of fluorescently-labeled anti-IgG under room temperature or 37 ℃ and hatched 1-2 hour, with the phosphate buffer mounting that contains 50% glycerine, observations under fluorescent microscope.
Described one anti-in incubation temperature be preferably 4 ℃; Described two anti-in incubation temperature be preferably 37 ℃, incubation time is preferably 1 hour.
Described gymnosperm can be lacebark pine, cdear, picea wilsonii, loose China fir guiding principle such as Picea meyeri gymnosperm; Be preferably the Pinaceae gymnosperm, be preferably lacebark pine and picea wilsonii especially.
Described phosphate buffer is with 10mM phosphate, 138mM sodium chloride, the preparation of 2.7mM potassium chloride.Method of the present invention can be with anti-this antigen of existing known antigens preparation one anti-, or directly obtain the anti-of this anti-known antigens from commercial channels, detect in the gymnospermae pollen pipe by Immunofluorescence Reactions and have or not this known antigens.
In the method for the present invention, paraformaldehyde can be fast fixing compositions such as pollen tube internal protein, DNA, plasma membrane, and do not destroy antigen.Lime chloride can be protected the stability of protoplast membrane.Cellulase can the enzymolysis endhymenine cellulose components, and components such as pectic substance in the macerozyme degraded endhymenine, thus can partly degrade inwall, Triton X-100 can further change cell membrane thoroughly simultaneously, makes the abundant combination of antigen-antibody.Because pollen tube is less, and it is attached on the microslide of poly-D-lysine bag quilt, can reach immobilized antigen, the purpose of handled easily.The phosphate buffer that use contains 50% glycerine carries out mounting, both can prevent the quick cancellation of fluorescence, simultaneously cheap saving material.The method that the present invention utilizes immunofluorescence technique to detect plant flowers tube cell endoantigen has overcome because pollen tube wall and obstacle that can't mark born of the same parents endoantigen, optimize various reaction conditionss simultaneously, antigen-antibody is in conjunction with abundant, mark is effective, highly sensitive, the visual signal to noise ratio (S/N ratio) height that scanning obtains can truly and fully reflect the distribution situation of antigen in pollen tube, has bigger actual application value.
Description of drawings
Fig. 1 is the location situation of observed G-protein on picea wilsonii pollen tube plasma membrane under laser confocal scanning microscope
Fig. 2 is the location situation of observed G-protein on lacebark pine pollen tube plasma membrane under laser confocal scanning microscope
Embodiment
Method among the following embodiment is conventional method if no special instructions.
Percent concentration in following examples if no special instructions, is mass percent concentration.
The detection of embodiment 1, picea wilsonii pollen tube endoantigen
Get the picea wilsonii pollen tube of cultivating 18h, suspend, make pollen tube suspending liquid, carry out the processing of following steps with sterilized water:
1) collect pollen tube, resuspended with pH7.2PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride), repeat this step then once;
2) add paraformaldehyde, making its final concentration is 2% (fresh preparation), and incubated at room 2h inhales and removes paraformaldehyde, uses the PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) of pH7.2 fully to wash 2 times, each 5min;
3) with the enzymolysis liquid (pH5.5, the 15mMMes that contain 1.5% cellulase cellulase R-10 (available from Yakult Honsha Co.Ltd) and 1.2% macerozyme macerozyme R-10 (available from Yakult Honsha Co.Ltd), 400mM sweet mellow wine, 5mM lime chloride and 1 μ gmL
-1Protease inhibitors (aprotinin, Calbiochem) enzymolysis picea wilsonii pollen tube is 10 minutes, and the pollen tube behind the enzymolysis is used pH7.2 immediately, 10mM phosphate buffer (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) washing; The PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) that adds the pH7.2 that contains 0.2%TritonX-100, use pH7.2PBS damping fluid (10mM phosphate behind the room temperature effect 5min, 138mM sodium chloride, 2.7mM potassium chloride) wash each 5min 4 times; Adjust cell concentration to 1 * 10
5Individual/ml, the pollen tube suspension is added drop-wise on the microslide of poly-D-lysine bag quilt, leaves standstill 10min under the room temperature, then with the pH7.2,10mM PBS damping fluid (the 10mM phosphate that contain 3% (3g/100ml) bovine serum albumin(BSA), 138mM sodium chloride, 2.7mM potassium chloride) at room temperature seal 1h;
4) hatched 12 hours at 4 ℃ with anti-G protein antibodies (Calbiochem) (the G protein antibodies being diluted to 20-50ug/ml) with the 10mM PBS that contains 1% bovine serum albumin(BSA); (contain PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) and 0.05%TritonX-100) clean 10min, totally 3 times with PBST.Exhaustion liquid, but can not be dry; (Sigma, St.Louis USA), are hatched 1h under 37 ℃, the samely clean 10min with PBST, blot liquid after cleaning 3 times to add fluorescein isothiocyanate (FITC) mark two anti-;
Dropwise 50 μ l sealing liquid (the PBS solution (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) that contains 50% (V/V) glycerine) on the pollen tube on the microslide, adds cover glass, observes, takes pictures under the air drying 30min, fluorescent microscope.
Establish negative control with respect to the picea wilsonii pollen tube: with the picea wilsonii pollen tube with immunity before animal blood serum replace anti-hatching, or omit one and anti-ly directly carry out two anti-hatching.The result shows that G-protein mainly is positioned on the picea wilsonii pollen tube plasma membrane, as shown in Figure 1 top concentration the highest (fluorescence is the brightest).And the negative control that is provided with is not observed fluorescence, illustrates that method of the present invention is reliable.
The detection of embodiment 2, lacebark pine pollen tube endoantigen
Get the lacebark pine pollen tube of cultivating 72h, suspend, make pollen tube suspending liquid, carry out the processing of following steps with sterilized water:
1) collect pollen tube, resuspended with pH7.2PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride), repeat this step then once;
2) add paraformaldehyde, making its final concentration is 4% (fresh preparation), and incubated at room 1h inhales and removes paraformaldehyde, uses the PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) of pH7.2 fully to wash 2 times, each 5min;
3) with the enzymolysis liquid (pH5.5, the 15mM Mes that contain 3% cellulase cellulase R-10 (available from Yakult Honsha Co.Ltd) and 2% macerozyme macerozyme R-10 (available from Yakult Honsha Co.Ltd), 400mM sweet mellow wine, 5mM lime chloride and 1 μ gmL
-1Protease inhibitors (pepstatin, Calbiochem) enzymolysis lacebark pine pollen tube is 8 minutes, and the pollen tube behind the enzymolysis is used pH7.2 immediately, 10mM phosphate buffer (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) washing; The PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) that adds the pH7.2 that contains 0.2%Triton X-100, use pH7.2PBS damping fluid (10mM phosphate behind the room temperature effect 5min, 138mM sodium chloride, 2.7mM potassium chloride) wash each 5min 4 times; Adjust cell concentration to 1 * 10
5Individual/ml, the pollen tube suspension is added drop-wise on the microslide of poly-D-lysine bag quilt, leaves standstill 10min under the room temperature, then with the pH7.2,10mM PBS damping fluid (the 10mM phosphate that contain 3% (3g/100ml) bovine serum albumin(BSA), 138mM sodium chloride, 2.7mM potassium chloride) at room temperature seal 1h;
4) hatched 12 hours at 4 ℃ with anti-G protein antibodies (Calbiochem) (the G protein antibodies being diluted to 20-50ug/ml) with the 10mM PBS that contains 1% bovine serum albumin(BSA); (contain PBS damping fluid (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) and 0.05%TritonX-100) clean 10min, totally 3 times with PBST.Exhaustion liquid, but can not be dry; (Sigma, St.Louis USA), are hatched 1h under 37 ℃, the samely clean 10min with PBST, blot liquid after cleaning 3 times to add fluorescein isothiocyanate (FITC) mark two anti-;
Dropwise 50 μ l sealing liquid (the PBS solution (10mM phosphate, 138mM sodium chloride, 2.7mM potassium chloride) that contains 50% (V/V) glycerine) on the bioplast on the microslide, adds cover glass, observes, takes pictures under the air drying 30min, fluorescent microscope.
Establish negative control with respect to the lacebark pine pollen tube: with the lacebark pine pollen tube with immunity before animal blood serum replace anti-hatching, or omit one and anti-ly directly carry out two anti-hatching.The result shows that G-protein mainly is positioned on the lacebark pine pollen tube plasma membrane, as shown in Figure 2 top concentration the highest (fluorescence is the brightest).And the negative control that is provided with is not observed fluorescence, illustrates that method of the present invention is reliable.
Claims (10)
1, a kind of method that detects gymnosperm pollen in-tube antigen may further comprise the steps:
1) with the phosphate buffer that contains paraformaldehyde with pollen tube fixing after, with the enzymolysis liquid enzymolysis gymnospermae pollen pipe of cellulase and macerozyme, be TritonX-100 or the NP-40 solution washing of 0.2%-1% again with mass percent concentration;
2) will carry out Immunofluorescence Reactions by the antibody corresponding with anti-determined antigen through the pollen tube that step 1) is handled, as detect fluorescence, then the gymnospermae pollen pipe contains this determined antigen and obtains the location situation of this determined antigen.
2, method according to claim 1 is characterized in that: in the described phosphate buffer that contains paraformaldehyde, the quality percentage composition of paraformaldehyde is 2-4%.
3, method according to claim 2 is characterized in that: the mass percent concentration of the paraformaldehyde that described fixedly pollen tube is used is 2%, and the set time is 2 hours.
4, method according to claim 1, it is characterized in that: the enzymolysis liquid of described cellulase and macerozyme contains the cellulase that mass percent concentration is 1.5-3%, mass percent concentration is the macerozyme of 1.2%-2%, pH5-6,15mM 2-morphine quinoline ethyl sulfonic acid, 400mM sweet mellow wine, 5mM lime chloride and 1 μ gmL
-1Protease inhibitors.
5, method according to claim 4 is characterized in that: the mass percent concentration of described cellulase is 1.5%, and the mass percent concentration of described macerozyme is 1.2%.
6, method according to claim 4 is characterized in that: the time of described enzymolysis gymnospermae pollen pipe is 8-15 minute, is preferably 10 minutes.
7, method according to claim 1 is characterized in that: in the described step 1), the mass percent concentration of TritonX-100 is 0.2%.
8, method according to claim 1, it is characterized in that: described step 2), it is that the pollen tube of handling through step 1) is transferred on the microslide of poly-D-lysine bag quilt that the described pollen tube antibody of handling through step 1) corresponding with anti-determined antigen carries out Immunofluorescence Reactions, at room temperature sealed 1-2 hour with bovine serum albumin solution, after phosphate buffer through containing 0.2-1%Triton X-100 or NP-40 fully washs, hatched 2-12 hour under 0-4 ℃ or the room temperature in anti-at one of anti-determined antigen, transfer in the two anti-solution of fluorescently-labeled anti-IgG under room temperature or 37 ℃ and hatched 1-2 hour, with the phosphate buffer mounting that contains 50% glycerine.
9, method according to claim 8 is characterized in that: described one anti-middle incubation temperature is 4 ℃; Described two anti-in incubation temperature be 37 ℃, incubation time is 1 hour.
10, method according to claim 1 is characterized in that: described gymnosperm is loose China fir guiding principle Pinaceae gymnosperm; Be preferably lacebark pine or picea wilsonii.
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| CN 200610003166 CN1811433B (en) | 2006-02-20 | 2006-02-20 | Method for detecting gymnosperm pollen in-tube antigen |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318679A (en) * | 2017-01-18 | 2018-07-24 | 中国农业大学 | A kind of immunofluorescence method and its kit of the woody Pollen Tubes albumen of positioning |
| CN113418766A (en) * | 2021-06-24 | 2021-09-21 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after whole immunization |
-
2006
- 2006-02-20 CN CN 200610003166 patent/CN1811433B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318679A (en) * | 2017-01-18 | 2018-07-24 | 中国农业大学 | A kind of immunofluorescence method and its kit of the woody Pollen Tubes albumen of positioning |
| CN113418766A (en) * | 2021-06-24 | 2021-09-21 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after whole immunization |
| CN113418766B (en) * | 2021-06-24 | 2023-10-03 | 中国科学院华南植物园 | Double-fixation embedding sample preparation method after integral immunization |
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