CN104165989A - Colloidal gold immunization test paper strip for detecting morchella mycelium - Google Patents

Colloidal gold immunization test paper strip for detecting morchella mycelium Download PDF

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Publication number
CN104165989A
CN104165989A CN201410327203.1A CN201410327203A CN104165989A CN 104165989 A CN104165989 A CN 104165989A CN 201410327203 A CN201410327203 A CN 201410327203A CN 104165989 A CN104165989 A CN 104165989A
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coated
antibody
test paper
hickory chick
line
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王一东
张强
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Changchun University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention relates to a colloidal gold immunization test paper strip for detecting morchella mycelium, and relates to improvement for production of edible fungi. The improvement is characterized in that a sample pad, a colloidal-gold pad, a cellulose nitrate membrane and absorbent paper are attached on a plastic liner plate in order, a colloidal gold-marked anti-common morel antibody is sprayed or immersed on the colloidal-gold pad, goat anti mouse IgG diluted by a PBS buffer is coated on a quality control line, the anti-common morel rabbit serum polyclonal antibody diluted by the PBS buffer is coated on a detection line, and a NC membrane is coated on the external surface of the quality control line and the detection line. The colloidal gold immunization test paper strip has the beneficial effect that the detection period is short (the result can be generated in 1 hour); operation is simple, professional staff and apparatus for detection are not required; cost is low (each detection sample costs a few yuan or more than ten yuan); and the qualitative detection result is accurate. The method can solve the problem that whether bacterial classification is common morel bacterial classification or not in artificial cultivation of common morel, and whether the mycelia grown in a cultivated culture material is common morel mycelia or not, and loss can be reduced.

Description

A kind of colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia
Technical field
The invention belongs to biochemical technology field, relate to a kind of improvement that edible fungi detects of producing.
Background technology
Hickory chick is a kind of rare edible fungi, and it is famous with delicious food.Research in recent years also finds that it has abundant health care and is worth.Because artificial cultivation technique is also immature, product leans on the collection of wild resource substantially, holds at high price.The artificial cultivation of realizing hickory chick has huge commercial value.We know, when hickory chick artificial cultivation, if the bacterial classification of cultivation is not hickory chick or nutrient culture media is by living contaminants in the course of cultivation, this situation can not cultivated hickory chick.In the artificial cultivation practice process of hickory chick, the Mining receipts of cultivating fructification (mushroom body) from bacterial classification approximately need 4 to 6 months, generally, only have fruiting could prove that the bacterial classification of inoculation is hickory chick bacterial classification, this is also traditional morphology calibration method, and the benefit of this authentication method is direct, objective; Maximum defect is excessive cycle, and because hickory chick cultivation, fruiting are subject to various factors to be difficult for fruiting, this method sheep is to being restricted in the early stage and interim detection of the artificial cultivation process of tripe bacterium.The method of another calibrating hickory chick is the DNA of the extraction hickory chick under laboratory condition, by nucleotide sequence, detect comparison and identify, the advantage of the method is precisely, and shortcoming is to need professional testing laboratory, sense cycle long (a couple of days), somewhat expensive.
List of references:
(1) Meng Meng, Cao Chi, Chen Hanzhong, etc. development and the Preliminary Applications [J] of the sick immunity colloidal gold test paper strip of Trypanosoma evansi in Buffalo. journal of animal science and veterinary medicine, 2011,42(7): 988-933.
(2) Wang Yidong, Liu builds. monoclonal antibody against toadstool and preparation method thereof: China, 200910218152.8[P] and .2010-06-09.
(3) Ma Shuai, Chen Hualin, Wang Yawen, etc. immunoenzymatic staining is to the Primary Location of Morciiella Esculeuta Mycelia specificity target position and analysis [J]. fungus research, and 2014,12(2): 115-118.
Summary of the invention
The object of the invention is: a kind of colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia is provided, and it can detect hickory chick easy, fast and accurately.
Technical scheme of the present invention is: on plastics lining board, be pasted with successively sample pad, gold mark pad, nitrocellulose filter, thieving paper, by on the anti-hickory chick antibody spraying of colloid gold label or immersion gold mark pad, to be coated on nature controlling line with the mountain sheep anti-mouse igg of PBS damping fluid dilution, by being coated on detection line with the anti-hickory chick rabbit anteserum polyclonal antibody of PBS damping fluid dilution, at nature controlling line and detection line appearance, be coated with NC film.
Mountain sheep anti-mouse igg is done the coated nature controlling line of 1/100,1/200,1/500,1/1000 and 1/2000 dilution with PBS damping fluid.
The anti-hickory chick rabbit anteserum polyclonal antibody that by concentration is 4.22 ug/ul is done 1,2,3,4,5 and 6 times of coated detection line of dilution with PBS damping fluid.
Preparation method of the present invention is:
(1) preparation of the anti-hickory chick antibody of colloid gold label (monoclonal antibody, polyclonal antibody)
Through the monoclonal antibody (C6A8 cell line) of ammonium sulfate precipitation method purifying and the protein concentration of rabbit polyclonal antibody, be respectively 2.91 ug/ul, 4.22 ug/ul.Tiring as 1:1280 of C6A8 monoclonal antibody, the tiring of rabbit anteserum: 1:2560.Colloidal gold solution pH is in 8.5 left and right, and C6A8 monoclonal antibody adds while making its final concentration be 0.58 ug/ml in colloidal gold solution, collaurum can be marked at preferably on anti-hickory chick monoclonal antibody C6A8.By the anti-hickory chick antibody of colloid gold label or spraying or immersion gold mark pad, by 37 ℃ of 1h of gold mark pad, dry, standby.
(2) the coated concentration of nature controlling line antibody and detection line antibody
Detection line coated antibody (use be that the anti-hickory chick of rabbit of purifying is how anti-) protein concentration is: 62.1ug/ml, nature controlling line coated antibody (mountain sheep anti mouse IgG(H+L)) protein concentration is: 6.4ug/ml.Mentioned reagent is coated with on nitrocellulose filter (NC film) correspondence position by the mode of spray or painting or point sample, and 37 ℃ of 1h, dry.The nitrocellulose membrane being coated with is soaked in to 1% the middle sealing of bovine serum albumin(BSA) (BSA), 37 ℃ of 1h, dry.Standby.
(3) assembling of test strips and test strips assay are judged.
As shown in Figure 1, successively sample pad, gold mark pad, nitrocellulose filter, thieving paper are affixed on plastics lining board, are cut into wide rectangular of 0.4cm;
Draw the appropriate sample to be checked through milled processed, be added drop-wise in the sample pad of test strips, visible sample is divided a word with a hyphen at the end of a line forward by chromatography effect, the red stripes that there will be naked eyes raised path between farm fields to see on NC film in the process of dividing a word with a hyphen at the end of a line, l0 min observations after application of sample, T line and C line all develop the color. and result is positive; The colour developing of C line, T line does not develop the color, and result is negative; If C line does not develop the color, test strips is invalid.
The invention has the beneficial effects as follows: there is sense cycle short (going out result in 1 hour); Easy and simple to handle, do not need professional and instrument and equipment to detect; Cost low (each sample cost needs several units to tens yuan); Qualitative detection result is accurate.This method can solve whether bacterial classification in hickory chick artificial cultivation is hickory chick bacterial classification, and cultivates whether the mycelia growing in rear compost is the monitoring of Morciiella Esculeuta Mycelia, can reduce the loss.
Accompanying drawing explanation
Fig. 1 is structural drawing of the present invention.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described:
Embodiment 1
As shown in the figure, the 1st, VC base plate P, the 2nd, sample pad, the 3rd, collaurum pad, the 4th, NC film, the 5th, absorbent filter, the 6th, nature controlling line, the 7th, detection line.
Hickory chick bacterial classification, numbering: 51589, be purchased from Chinese agriculture microorganism fungus kind preservation center;
The anti-hickory chick monoclonal antibody in mouse source (C6A8 hybridoma cell strain), the anti-hickory chick polyclonal antibody in/rabbit source, the preparation of Ji You Changchun University of Science and Technology Life Sci-Tech institute bioengineering dept laboratory, anti-hickory chick monoclonal antibody hybridoma cell strain is now stored in this laboratory;
Colloidal gold solution, Jin Biao bio tech ltd, Shanghai;
Gold mark pad, sample pad, thieving paper, cellulose nitrate (NC) film, Jin Biao bio tech ltd, Shanghai;
Bovine serum albumin(BSA) (BSA), the packing of sigma company;
HRP mark mountain sheep anti mouse IgG(H+L), HRP mark goat anti-rabbit igg (H+L) is purchased from Beijing biotechnology Ltd of Zhong Shan Golden Bridge;
PH test paper, the magnificent company in Shanghai;
Test required key instrument, equipment: the preparation of Milli-Q ultrapure water system, magnetic stirring apparatus, constant incubator, refrigerated centrifuge, microplate reader, etc.
1 method
The processing of 1.1 reagent preparations and glassware
(1) 0.1% solution of potassium carbonate: accurately take 0.1 g sal tartari+ultrapure water 100 ml and be mixed with solution;
(2) PBS damping fluid (0.01 mol/L phosphate buffer): accurately take sodium chloride 8 g, potassium chloride 0.2 g, potassium dihydrogen phosphate 0.2 g, sodium hydrogen phosphate 2.9 g, adding distil water is settled to 1000 ml;
(3) envelope antigen dilution; 0.01 mol/L PBS pH7.4;
(4) resuspended liquid (containing the PB liquid of 1%BSA): by the sodium hydrogen phosphate 9.47mL of the sodium dihydrogen phosphate of 0.2 mol/L, 0.53 ml and 0.2mol/L, after mixing, add BSA to final concentration be 1%.
Glassware used first cleans up with detersive, then with hydrochloric acid lotion, soaks 24 h, then washes 3～4 times with distilled water immersion 24h, last distilled water, distilled water, standby after drying [1].
The purifying of 1.2 monoclonal antibodies and tiring and the mensuration of protein concentration
Use ammonium sulfate precipitation method [2]monoclonal antibody C6A8 and the polyclonal antibody of the anti-hickory chick of difference purifying.Antibody after purifying is measured it by ELISA method respectively and is tired, and antibody dilution 20,40,80......5120 are doubly added respectively to 1-9 hole, and 10,11 hole SP2/0 are as negative control.Coomassie brilliant blue method is surveyed its protein concentration.
Determining of 1.3 colloid gold label optimal pHs
With the sal tartari of 0.1mol/L and the hydrochloric acid of 0.1mol/L, colloidal gold solution is adjusted to different gradients, get pH and be each 100ul of colloidal gold solution of 7,8,8.5,9,10, add respectively 1ul concentration for the good antibody of purifying of mg/mL l.14, mix, under room temperature, place 15 min, adding respectively 2ul concentration is 10% NaCl solution, mixes, and under room temperature, places 3h.Unaccommodated pH value group solution colour becomes blueness from redness, and even blackening, colourless is observed the group of change color minimum for to stablize group, and this pH value is the suitableeest mark potential of hydrogen.
1.4 colloid gold labels are determining of the suitableeest protein concentration
With the sal tartari of 0.1mol/L and the hydrochloric acid solution of 0.1mol/L, colloidal gold solution is adjusted to optimal pH, in every 100ul colloidal gold solution, add respectively 1.2,0.9,0.6,0.3,0.l, 0.05ul monoclonal antibody, room temperature effect 5 min, add respectively 10% NaCl solution 5ul, mix rear standing 3h [4].Observe the less group of change color, record adds the amount of monoclonal antibody, and this albumen two is the suitableeest label concentration.
The preparation of 1.5 colloidal gold labeled monoclonal antibodies and detection
Get 100uL colloidal gold solution, at the uniform velocity, under beating action, add the sal tartari of appropriate 0.1mol/L, regulate colloidal gold solution to optimal pH, monoclonal antibody solution is dropwise added in colloidal gold solution, about 5 min add, even
Speed stirs 30 min; Room temperature is placed 15 min, dropwise add 10%BSA to final concentration be 1%, the colloidal gold solution of mark, with centrifugal 20 min of 3 000r/min, is discarded to precipitation.Centrifugal 60 min of 12 000 r/min, inhale and abandon supernatant; With the abundant dissolution precipitation of PB liquid containing 1% bovine serum albumin(BSA).4 ℃ save backup [5].
In order to detect monoclonal antibody, whether be combined upper with collaurum, we adopt direct method, be about to positive antigen and mountain sheep anti mouse IgG(bis-anti-) be soaked in the colloidal gold solution of labelled antibody after being coated in respectively on nitrocellulose membrane, after 20 minutes, remove nitrocellulose membrane and observe its colour developing result.
Determining of the coated concentration of 1.6 nature controlling line antibody and detection line antibody
Existing mountain, laboratory sheep anti-mouse igg is done to the coated nature controlling line of 1/100,1/200,1/500,1/1000 and 1/2000 dilution with PBS damping fluid.Be soaked in the colloidal gold solution of labelled antibody, after 20 minutes, observe its colour developing situation, select to be applicable to the coated NC film of concentration.
Determine after two anti-coated concentration, the anti-hickory chick rabbit anteserum polyclonal antibody that by concentration is 4.22 ug/ul is done 1,2,3,4,5 and 6 times of coated detection line of dilution with PBS damping fluid, assembling test strips, do positive and negative sample detection, observe colour developing situation, select the coated NC film of antibody of optium concentration.
The sealing of 1.7 detection lines and nature controlling line
By the coated concentration of the suitableeest detection line and nature controlling line, be coated in after nitrocellulose membrane, the nitrocellulose membrane be coated be soaked in 1% bovine serum albumin(BSA) and seal, be positioned in 37 degree constant temperature ovens taking-up after a hour, oven dry.
The assembling of 1.8 test strips
Successively sample pad, gold mark pad, nitrocellulose filter, thieving paper are affixed on plastics lining board, are cut into 0.4cm
Wide is rectangular; Assigned address at nitrocellulose filter is coated with certain density mountain sheep anti-mouse igg (nature controlling line) and rabbit anteserum polyclonal antibody (detection line) point sample.
1.9 test strips assays are judged
Drawing appropriate sample drop to be checked is added in the sample pad of test strips, visible sample is divided a word with a hyphen at the end of a line forward by chromatography effect, the red stripes that there will be naked eyes raised path between farm fields to see on NC film in the process of dividing a word with a hyphen at the end of a line, l0 min observations after application of sample, T line and C line all develop the color. and result is positive; The colour developing of C line, T line does not develop the color, and result is negative; If C line does not develop the color, test strips is invalid.
2 experimental results
Monoclonal antibody and many anti-tiring and the mensuration of protein concentration after 2.1 purifying
It is that tiring of 320, C6A8 is 1280 that anti-hickory chick monoclonal antibody W8C9, C6A8 after purifying and polyclonal antibody rabbit anteserum record tiring of W8C9 by ELISA indirect method, and tiring of rabbit anteserum is 2560.
With Coomassie brilliant blue method bovine serum albumin typical curve in vain, record monoclonal antibody W8C9, C6A8 and polyclonal antibody rabbit anteserum protein concentration is respectively: 2.28 ug/ul, 2.91 ug/ul, 4.22 ug/ul.
Due to C6A8 after purifying tire or higher than the tiring of W8C9, so C6A8 labeling of monoclonal antibody collaurum is selected in this experiment.
The suitableeest flag condition of 2.2 collaurums
Through observation and comparison, during colloidal gold solution pH=8.0, its change color is minimum, so determine that colloid gold label optimal pH is 8.0.Experimental result shows, antibody is added to best results while making its final concentration be 0.58 ug/ml in colloidal gold solution, so 0.58 ug/ml is selected in this experiment, is optimum antibody label concentration.
The result of 2.3 colloid gold labels
After in the nitrocellulose membrane that is coated with positive antigen the has been soaked in mark colloidal gold solution of antibody, there is obvious red appearance in the place of observing envelope antigen, instruction book clonal antibody in antibody in conjunction with upper.
Determining of the coated concentration of 2.4 the suitableeest nature controlling lines
While determining sheep anti mouse two anti-1/500 dilution of nature controlling line mountain through experiment, experiment effect is best.
2.5 specificity results
We are coated in nitrocellulose membrane (detection line) by above-mentioned ten kinds of different positive mycelia antigen samples that detect with the ELISA sandwich method of setting up, mountain sheep anti mouse IgG(bis-is anti-) coated as nature controlling line, then be soaked in the colloidal gold solution that indicates antibody, after 20 minutes, observe, result is as following table:
The experiment of table 2-1 colloidal gold method specificity
Note: 1. sample is that difference of the same race and not of the same race is cultivated the Morciiella Esculeuta Mycelia treating fluid of (liquid) batch.
2. the disposal route of sample is, goes appropriate mycelia, adds appropriate aseptic sea sand, grinds 20 minutes in mortar, and supernatant is.
Visible, the Morciiella Esculeuta Mycelia antigen sample that can detect with double antibody ELISA sandwich method also can detect its positive with colloidal gold method, and colloidal gold immunity chromatography is consistent with double antibody ELISA sandwich method testing result.
And adopt this method to detect auricularia auriculajudae, asparagus, gold oyster mushroom, mushroom, flat mushroom, the pholiota nameko of variable concentrations, during the hypha of edible fungus treating fluid of two spore mushrooms, its result is identical with negative control value, shows that the method has good specificity to the detection of Morciiella Esculeuta Mycelia antigen.
3 test strips using method
The processing of 3.1 samples: get the bacterial classification that contains doubtful Morciiella Esculeuta Mycelia of appropriate (1g left and right) or the compost sample (can be mixed with nutrient culture media) that contains doubtful Morciiella Esculeuta Mycelia, add the aseptic sea sand of about 2g and appropriate pure water (can with clean commercially available mineral water), in mortar, grind 1020 minutes, standing 5 minutes, the limpider part in top of liquid can be used as sample.
3.2 detect: sample is splashed in the sample pad of test strips 1 to 2 (or in sample well of the test strips of assembling), under room temperature standing 20 minutes, observe testing result.
The judgement of 3.3 results:
If all there is red bar line in detection line (from adding close to sampling point) and nature controlling line (from adding away from sampling point), judge that sample is positive, both in sample, contained hickory chick;
If only have nature controlling line to occur red bar line, judge that sample is negative, both in sample, do not contained hickory chick;
If detection line and nature controlling line all do not occur or only have detection line to occur red bar line, be judged to be test strips and lost efficacy.

Claims (4)

1. a colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia, it is characterized in that: on plastics lining board, be pasted with successively sample pad, gold mark pad, nitrocellulose filter, thieving paper, by on the anti-hickory chick antibody spraying of colloid gold label or immersion gold mark pad, to be coated on nature controlling line with the mountain sheep anti-mouse igg of PBS damping fluid dilution, to also be coated on detection line with the anti-hickory chick rabbit polyclonal antibody of PBS damping fluid dilution, in the PBS damping fluid by the NC film that is coated with nature controlling line and detection line through containing 1% bovine serum albumin(BSA), seal.
2. a kind of colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia as claimed in claim 1, it is characterized in that: colloidal gold solution pH is 8.0 8.5, anti-hickory chick monoclonal antibody C6A8 adds while making its final concentration be 0.58 ug/ml in colloidal gold solution, by colloid gold label on anti-hickory chick monoclonal antibody C6A8.
3. a kind of colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia as claimed in claim 1, is characterized in that: it is the coated nature controlling line of 6.4ug/ml that mountain sheep anti-mouse igg is diluted to protein concentration with PBS damping fluid.
4. a kind of colloid gold immune test paper bar that detects Morciiella Esculeuta Mycelia as claimed in claim 1, is characterized in that: will with PBS damping fluid, be diluted to the coated detection line of anti-hickory chick rabbit polyclonal antibody that protein concentration is 62.1ug/ml.
CN201410327203.1A 2014-07-10 2014-07-10 Colloidal gold immunization test paper strip for detecting morchella mycelium Pending CN104165989A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113484516A (en) * 2021-07-01 2021-10-08 山东和生菌业科技有限公司 Quantitative detection method for morchella mycelium

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CN113484516A (en) * 2021-07-01 2021-10-08 山东和生菌业科技有限公司 Quantitative detection method for morchella mycelium

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Application publication date: 20141126