CN103792360A - Preparation and detection method of enzyme-linked immunosorbent assay kit for aspergillus versicolor - Google Patents

Preparation and detection method of enzyme-linked immunosorbent assay kit for aspergillus versicolor Download PDF

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CN103792360A
CN103792360A CN201210435692.3A CN201210435692A CN103792360A CN 103792360 A CN103792360 A CN 103792360A CN 201210435692 A CN201210435692 A CN 201210435692A CN 103792360 A CN103792360 A CN 103792360A
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antibody
sterigmatocystin
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杜道林
祝文珍
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Jiangsu Wise Science and Technology Development Co Ltd
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Abstract

The invention discloses a special kit for enzyme-linked immunosorbent assay of aspergillus versicolor, which has the advantages of fast, sensitive and accurate detection, quantification, simplicity ad convenience in operation, low requirement on the sample purity and strong specificity and is particularly suitable for mass samples. Therefore, the invention also provides a preparation and detection method of the special kit. The special kit comprises a washing solution, a color developing solution and a stop solution and is characterized by comprising a coating plate coating a solid-phase antigen against aspergillus versicolor, a standard product of aspergillus versicolor (ST), a monoclonal antibody against aspergillus versicolor and an enzyme-labeled antibody against aspergillus versicolor.

Description

Enzyme-linked immunosorbent assay kit preparation and the detection method of sterigmatocystin
Technical field
The present invention relates to biological technical field, be specially the preparation and the specialty detection that sterigmatocystin Enzyme-linked Immunosorbent Assay are detected to dedicated kit.
Background technology
Sterigmatocystin (ST) is mainly the final metabolic product of aspergillus versicolor and aspergillus nidulans, is again that aspergillus flavus and aspergillus parasiticus generate the yellow blood intermediate product in mould toxin process later stage simultaneously.It is reported, infected the corn of aspergillus versicolor under the environment of 27 ℃, more than within 21 days, can producing sterigmatocystin 12 g/kg.Sterigmatocystin is first isolated in by aspergillus versicolor culture in 1954, but does not cause people's enough attention, to finding after the strong carcinogenicity of aflatoxin, just starts to note.Confirm that by 14 ~ labeling method sterigmatocystin can be transformed into aflatoxin horse, and had people to think that sterigmatocystin may be the cause of disease of African certain areas liver cancer.
The bacterial classification that can produce ST is mainly aspergillus versicolor, aspergillus nidulans and Bipolaris hawaiiensis.In addition, Aspergillus chevalieri, red aspergillus, aspergillus ustus, Amsterdam aspergillus, yellowish-brown aspergillus, four ridge aspergillus, variable color aspergillus, pawl aspergillus, chactomium globosum and aspergillus flavus and aspergillus parasiticus etc. also can produce ST.Above-mentioned these moulds are extensively present in nature, can pollute grain, food and the forage grasses such as barley, wheat, corn, peanut, soybean, coffee bean, ham, cheese, especially pollute even more serious to the feeds such as wheat, corn, peanut and forage grass.What the poison amount of producing was the highest is aspergillus versicolor, is secondly aspergillus nidulans and Bipolaris hawaiiensis, and the amount that the former produces ST is about 2 times of the latter.
The present invention is that the Enzyme-linked Immunosorbent Assay of sterigmatocystin detects dedicated kit.Its detection is quick, sensitive, accurate, can be quantitative, easy and simple to handle, and less demanding to sample purity, high specificity, is specially adapted to the detection of batch samples.
Summary of the invention
The invention provides preparation and the detection method of dedicated kit.Comprising cleansing solution, nitrite ion, stop buffer.It is characterized in that: be surrounded by coated plate, sterigmatocystin (ST) standard items of sterigmatocystin solid phase antigen, sterigmatocystin monoclonal antibody, sterigmatocystin enzyme labelled antibody.
When detection, get coated plate, add standard items/sample 50 μ L in corresponding micropore, then add enzyme labeling thing 50 μ L/ holes, and then add 50 μ L/ hole antibody working fluids, vibration mixes gently, reacts 30 min by cover plate membrane cover plate postposition room temperature lucifuge.Carefully open cover plate film, liquid in hole is dried, add cleansing solution 250 μ L/ holes, soak 15 ~ 30 s at every turn, fully wash 4 ~ 5 times, pat dry with thieving paper.Add nitrite ion 100 μ L/ holes, vibration mixes gently, with rearmounted 25 ℃ of lucifuge environment reaction 15 min of cover plate membrane cover plate.Every hole adds 50 μ L stop buffers, sets microplate reader at 450 nm places, measures OD value (suggestion 450/630 nm dual wavelength detects, and runs through data in 5 min).The content of ST in calculation sample from typical curve.The mean value (B) of the titer recording or sample absorbance is divided by the absorbance (B of first titer (0 titer) 0) value is multiplied by 100% again, obtains percentage absorbance,
Percentage absorbance (%)=B/B 0× 100%
Take the logarithm at 10 end of as of standard items concentration as X-axis, percentage light absorption value is Y-axis, drawing standard curve.By the percentage light absorption value substitution typical curve of sample, read the corresponding value of sample from typical curve, as 10 power, be multiplied by extension rate, be the amount of contained sterigmatocystin in sample.
Accompanying drawing explanation
Fig. 1 is sterigmatocystin canonical plotting.
Embodiment
Embodiment 1 Sample pretreatment method
Accurately take 5.00 g samples, add 45 mL acetonitriles, after 40% sulfuric acid solution that 5 mL contain 4%KCl, 30 min that vibrate on vibrating machine incline and clear liquid, wash at twice residue with 10 mL acetonitriles again, collect acetonitrile liquid and merge with said extracted liquid, (sherwood oil consumption is 20 to the petroleum ether degreasing three for extract merging, 15, 10 mL), extract after the degreasing 25mL that adds water, with chloroform extraction, three times (chloroform consumption is 20, 15, 10mL), the chloroform layer of collecting is lower freezing at low temperature (6 ℃), then filter deicing, degrease, with rotary evaporator evaporate to dryness (40 ℃ of temperature are following), residue dissolves rear for chromatographic determination with sample diluting liquid.
The enzyme-linked immunosorbent assay kit preparation of embodiment 2 sterigmatocystins
1, the coated plate of sterigmatocystin solid phase antigen is made, comprise coating antigen making sealing, liquid configuration and hatch.The wrapper sheet that adds coating buffer to be used as solid phase antigen coating antigen is made.Coating buffer: 1.6 g sodium carbonate add 2.9 g sodium bicarbonates, add distilled water to 1000 mL, regulates pH to 9.6.Confining liquid: 1 gBSA, 100mLPBS.Every hole 100 μ L wrapper sheet liquid when wrapper sheet, hatch 2 h at 37 ℃, and plate twice is washed in rear taking-up, adds confining liquid, and every hole 180 μ L are hatched 1.5 h at 37 ℃, take out and dry, and add 2 ~ 8 ℃ of preservations of drying agent;
2, the coupling of coating antigen:
Carbodiimide (EDC) method coupling sterigmatocystin haptens and protein carrier: get 5 mg sterigmatocystin haptens and 2.5 mg cBSA and be dissolved in 250 μ L TE buffer(pH 8.0) in, fully concussion is dissolved it, then gets EDCHCl 79 mg and be fully dissolved in 250 μ LTE buffer(pH 8.0) in.The concussion limit, mixed solution limit of sterigmatocystin haptens and cBSA is dropwise added to EDC solution, react 2 h in 37 ℃ of shaking tables more than.First use Sephadex post separation coupling product and the little molecule of unconjugated sterigmatocystin haptens, then the purifying of dialysing, by 0.5 mL solution, 3 d that dialyse, change liquid twice every day in PBS, change liquid 200 mL at every turn.The low dose of packing of dialysis product sterigmatocystin hapten conjugation albumen, in-20 ℃ of preservations, for subsequent use;
3, sterigmatocystin method for preparing monoclonal antibody:
I materials and methods
(1) reagent
Artificial antigen: sterigmatocystin-bovine serum albumin(BSA) compound, Freund's complete adjuvant and incomplete Freund's adjuvant, Glu, bovine serum albumin(BSA) Part V, PEG-6000,2,6,10,14-tetramethyl-pentadecane (Pristane), o-phenylenediamine, dimethyl sulfoxide (DMSO), Tris, Hepes pH buffering agent, immunoglobulin (Ig), horseradish peroxidase mark thing, mouse IgG and goat anti-mouse igg etc., other conventional chemical reagent is that top grade is pure, analytical reagent;
(2) immune animal
8 week age be the capable secondary immunity of artificial antigen sterigmatocystin-BSA for BALB/c male mice; Every mouse fundamental immunity dosage is 100 μ g, lumbar injection: booster immunization after 24d, and tail vein injection, gets spleen and merges after 4 d;
(3) Fusion of Cells
Immune mouse spleen cell mixes with 10:1 with myeloma cell Sp-2/0.Be that 1000 polyglycol are made fusion agent with 50% molecular weight, after fused cell suspends with the HAT selective medium containing 20% calf serum, be inoculated in like Turnover of Mouse Peritoneal Macrophages and do in 96 well culture plates of feeder layer, in 50% CO2, under 37 ℃ of conditions, cultivate, after 7 d, every culture hole is changed 2/3HT nutrient solution.After 9 d, start microscopy to have the culture hole of hybridoma clonal growth, get supernatant and screen, the cell that testing result is positive carries out cloning with limiting dilution assay immediately;
(4) ascites antibody purifying
Purify ascites antibody with ammonium sulfate salting-out process;
(5) hybridoma screening and antibody test
Hybridoma screening is undertaken by solid phase antigen indirect competition ELISA method: the concentration of envelope antigen sterigmatocystin-BSA is 15 μ g/mL, every hole dosage 120 μ L; The sterigmatocystin (10 μ g/mL) that the contained antibody antigen reactant in every hole is demarcated concentration by 80 μ L culture supernatant+2 μ L through ultraviolet spectrophotometer forms; The antigen part detecting in control wells uses 20 μ L toxin dilutions (20%MeOH-PBS) to replace; O-phenylenediamine for zymolyte (OPD); Other is undertaken by standard method;
(6) characteristic of antibody
1. antibody titer:
Measure with the indirect noncompetitive ELISA of solid phase antigen, test condition is: envelope antigen is sterigmatocystin-BSA, concentration 15 μ g/mL, every hole dosage 150 μ L: antibody is since 100 times of two-fold dilutions, every hole dosage 130 μ L; Antibody diluent is 0.1% BSA-PBS; SP-2/0 myeloma cell's culture supernatant for negative control hole; Confining liquid is 1%BSA-PBS, every hole dosage 250 μ L; Zymolyte OPD.Other is undertaken by standard method;
2. examination criteria toxin sensitivity:
First filter out the optimal combination of envelope antigen concentration and antibody working dilution by chessboard titrimetry, as test condition, the standard of making sterigmatocystin by solid phase antigen indirect competition ELISA method suppresses curve, and result is carried out to Mathematical Statistics Analysis.Wherein ELISA test condition is: the concentration of coated coating antigen sterigmatocystin-BSA coupling protein is 5 μ g/mL, every hole dosage 150 μ L; Antibody working fluid dilution ratio is 1:25600; Antibody diluent is 0.1% BSA-PBS; Antibody antigen reactant liquor is every hole 65 μ L antibody+65 μ L respective concentration sterigmatocystins; Confining liquid is 1%BSA-PBS, every hole dosage 250 μ L; Zymolyte is OPD; Sp-2/0 myeloma cell's culture supernatant for negative control.Other is undertaken by standard method;
3. the specificity of antibody:
Measure by solid phase antigen indirect competition ELISA method, the toxin of participating in the experiment is sterigmatocystin, aspergillus nidulans, and aspergillus flavus, aspergillus parasiticus, aflatoxin horse, other test condition is same 2.;
4. Subclass of antibody analysis:
Carry out with immune double diffusion method;
5. the affinity of antibody:
Friguet method.The titration of corresponding mouse IgG typical curve goes out IgG antibody content, corresponding antibody titer curve is obtained under equilibrium state again, free antibodies molar concentration ［ Ab ］ when antibody makes antigen reach semi-saturation, substitution following formula is obtained the affinity costant K (L/moL) of antibody
Ka＝１/［Ab］
(7) sterigmatocystin extraction and purification in sample;
II result and discussion
After the screening Fusion of Cells of Fusion of Cells one and hybridoma, grow hybridoma clone in approximately 57.6% hole, wherein antibody test positive rate is about 88%.Therefrom select the original strong inhibiting hole of antagonism, through subclone repeatedly, set up 5 anti-sterigmatocystin monoclonal antibodies of stably excreting the each cell line of hybridoma cell strain after twice subclone, positive rate has reached 100 %.2H8 has repeatedly (doubting is false bright property) when the 3rd time cloning, so carried out subclone 4 times.By above-mentioned antibody purification.
4, the standard items of sterigmatocystin are formed by high standard dilution, and dilution is PBS.In 0.1M PBS:1000 mL distilled water, add NaCl 9 g, Na 2hPO 412H 2o 6g, NaH 2pO 42H 2o 0.4 g, pH:7.2.
5, the enzyme of the enzyme labelled antibody of sterigmatocystin is by horseradish peroxidase mark.This enzyme labelled antibody is made of cross-linking method, and connection agent is 25% glutaraldehyde water solution.Principle is to utilize on glutaraldehyde molecule two symmetrical aldehyde radicals, respectively with enzyme and protein molecule in free amino, phenolic group etc. carry out mark with covalent bonds.When mark, should adopt fresh sterling, as the OD of the eleventh of the twelve Earthly Branches dialdehyde as far as possible 235nm/OD 280when nm < 3, the crosslinking chemical being.This invention adopts single stage method to make, and horseradish peroxidase, glutaraldehyde is added and is cross-linked together with aspergillus versicolor antibody.Then give excessive glutaraldehyde and prepare the enzyme conjugates with high molecular.Due to antibody molecule amount large (160,000), enzyme molecular weight little (40,000), the amino number of antibody molecule is more much more than zymoprotein molecules of ammonia radix, and antibody-glutaraldehyde that result generates or antibody-glutaraldehyde-antibody are many and cause the activity of enzyme mark thing little.Single stage method operation:
1. the preparation of anti-IgG-sterigmatocystin: 10 mg sterigmatocystins are dissolved in 1 mLPBS (0.01 moL/L pH7.0) that contains the anti-IgG of 5 mg, in ice bath, slowly stir, avoid Bubble formation as far as possible, after dissolving completely, slowly drip 1% glutaraldehyde solution 4 ml, the ultimate density that makes glutaraldehyde is 0.2%, move in room temperature and leave standstill after 2h ~ 3h, fully dialyse or remove excessive glutaraldehyde with SephadexG25 post with 4 ℃ of 0.01 moL/L pH7.0 PBS liquid, 4 ℃ save backup (also can be stored in the mid-low temperature refrigerator of 50% glycerine); 2. the preparation of anti-IgG-HRP: 12 mgHRP are dissolved in containing in 1 mLPBS (0.1mol/L pH6.8) of the anti-IgG of 5 mg, add 1% glutaraldehyde solution 4 mL under slowly stirring, put room temperature 2h ~ 3h, fully dialysis or with SephadexG 25except glutaraldehyde, after packing, be stored in low temperature refrigerator in a small amount, avoid multigelation, or freeze drying is preserved.
6, the preparation of cleansing solution:
Ten hydrogen phosphate dihydrate sodium 68.8 g add sodium dihydrogen phosphate 6.9 g, sodium chloride 45 g, and Tween-20 0.5 mL, adds distilled water to 1L, regulates pH to 7.4.
7, the preparation of nitrite ion:
Nitrite ion A: anhydrous sodium acetate 8.2 g, powder-beta-dextrin 2.5 g, carbamide peroxide 428.6 g, add distilled water to 1 L, regulate pH to 5.0, and 4 ℃ of preservations, reach room temperature when use.Nitrite ion B:100 mgTMB is dissolved in 10 mLDMSO, and brown bottle is preserved.When use, 14.6 mL nitrite ion A are mixed 15 minutes with 0.45 mL nitrite ion B.
8, stop buffer preparation:
2M sulfuric acid: the concentrated sulphuric acid thin up of 0.11 L 18M is to 1 L.
Embodiment 3 determination steps:
1, required reagent and microwell plate are taken out from cold storage environment, at room temperature balance 30 min, must shake up before every kind of liquid uses.Notice that titer all needs to do 2 parallel experiments;
2, add standard items: add standard items/sample 50 μ L in corresponding micropore, then add enzyme labeling thing 50 μ L/ holes, and then add 50 μ L/ hole antibody working fluids, vibration mixes gently, react 30 min by cover plate membrane cover plate postposition room temperature lucifuge;
3, wash plate: carefully open cover plate film, liquid in hole is dried, add cleansing solution 250 μ L/ holes, soak 15 ~ 30 s at every turn, fully wash 4 ~ 5 times, pat dry with thieving paper;
4, colour developing: add nitrite ion 100 μ L/holes, vibration mixes gently, with rearmounted 25 ℃ of lucifuge environment reaction 15 min of cover plate membrane cover plate;
5, measure: every hole adds 50 μ L stop buffers, set microplate reader at 450 nm places, measure OD value (suggestion 450/630 nm dual wavelength detects, and runs through data in 5 min);
6, interpretation of result :
The mean value (B) of measured titer or sample absorbance is divided by the absorbance (B of first titer (0 titer) 0) value is multiplied by 100% again, obtains percentage absorbance,
Percentage absorbance (%)=B/B 0× 100%
Take the logarithm at 10 end of as of standard items concentration as X-axis, percentage light absorption value is Y-axis, drawing standard curve.By the percentage light absorption value substitution typical curve of sample, read the corresponding value of sample from typical curve, as 10 power, be multiplied by extension rate, be the amount of contained sterigmatocystin in sample.

Claims (6)

1. the preparation of sterigmatocystin kit, comprises cleansing solution, nitrite ion, stop buffer; It is characterized in that: be surrounded by coated plate, sterigmatocystin (ST) standard items of assorted aspertoxin solid phase antigen, sterigmatocystin monoclonal antibody, sterigmatocystin enzyme labelled antibody.
2. the coated plate of sterigmatocystin solid phase antigen is made, comprise coating antigen making, confining liquid configuration and hatch; The coupling of coating antigen: as follows according to the architectural feature coupling carrier protein B SA method of aspergillus versicolor:
Carbodiimide (EDC) method coupling sterigmatocystin haptens and protein carrier; Get 5 mg sterigmatocystin haptens and 2.5 mg cBSA and be dissolved in 250 μ LTE buffer(pH 8.0) in, fully concussion is dissolved it, then gets EDCHCl 79 mg and be fully dissolved in 250 μ L TE buffer(pH 8.0) in; The concussion limit, mixed solution limit of sterigmatocystin haptens and cBSA is dropwise added to EDC solution, react 2 h in 37 ℃ of shaking tables more than; First use Sephadex post separation coupling product and the little molecule of unconjugated sterigmatocystin haptens; The purifying of dialysing again, by 0.5 mL solution, 3 d that dialyse in PBS, changes liquid twice every day, changes liquid 200 mL at every turn; The low dose of packing of sterigmatocystin conjugate dialysis product making, in-20 ℃ of preservations, for subsequent use; The wrapper sheet that coating antigen adds coating buffer to be used as solid phase antigen is made; Coating buffer: 1.6 g sodium carbonate add 2.9 g sodium bicarbonates, add distilled water to 1000 mL, regulates pH to 9.6; Confining liquid: 1 g BSA, 100 mL PBS; Every hole 100 μ L wrapper sheet liquid when wrapper sheet, hatch 2 h at 37 ℃, and plate twice is washed in rear taking-up, adds confining liquid, and every hole 180 μ L are hatched 1.5 h at 37 ℃, take out and dry, and add 2 ~ 8 ℃ of preservations of drying agent.
3. according to described in claims Article 1, sterigmatocystin method for preparing monoclonal antibody
I materials and methods
(1) reagent
Artificial antigen: sterigmatocystin-bovine serum albumin(BSA) compound, Freund's complete adjuvant and incomplete Freund's adjuvant, Glu, bovine serum albumin(BSA) Part V, PEG-6000,2,6,10,14-tetramethyl-pentadecane (Pristane), o-phenylenediamine, dimethyl sulfoxide (DMSO), Tris, Hepes pH buffering agent, immunoglobulin (Ig), horseradish peroxidase, mouse-anti IgG etc., other conventional chemical reagent is that top grade is pure, analytical reagent;
(2) immune animal
8 week age, BALB/c male mice carried out secondary immunity with artificial antigen sterigmatocystin-BSA; Every mouse fundamental immunity dosage is 100 μ g lumbar injections; Booster immunization after 24 d, dosage is every mouse 100 μ g, tail vein injection is got spleen and is merged after 4 d;
(3) Fusion of Cells
Immune mouse spleen cell mixes with 10:1 with myeloma cell Sp-2/0;
Make fusion agent with the polyglycol that 50% molecular weight is 1000, after fused cell suspends with the HAT selective medium containing 20% calf serum, being inoculated in similar Turnover of Mouse Peritoneal Macrophages does in 96 well culture plates of feeder layer, in 50% CO2, under 37 ℃ of conditions, cultivate after 7 d, every culture hole is changed 2/3HT nutrient solution; After 9 d, start microscopy to have the culture hole of hybridoma clonal growth, get supernatant and screen, the cell that testing result is positive carries out cloning with limiting dilution assay immediately;
(4) ascites antibody purifying
Purify ascites antibody with ammonium sulfate salting-out process;
(5) hybridoma screening and antibody test
Hybridoma screening is undertaken by solid phase antigen indirect competition ELISA method: the concentration of envelope antigen sterigmatocystin-BSA is 15 μ g/mL, every hole dosage 120 μ L; The sterigmatocystin (10 μ g/mL) that the contained antibody antigen reactant in every hole is demarcated concentration by 80 μ L culture supernatant+2 μ L through ultraviolet spectrophotometer forms; The antigen part detecting in contrast uses 20 μ L toxin dilutions (20%MeOH-PBS) to replace; O-phenylenediamine for zymolyte (OPD); Other is undertaken by standard method;
(6) characteristic of antibody
1. antibody titer:
With solid phase antigen, indirect noncompetitive ELISA measures, and test condition is: envelope antigen is sterigmatocystin-BSA, concentration 15 μ g/mL, every hole dosage 150 μ L; Antibody is since 100 times of two-fold dilutions, every hole dosage 130 μ L; Antibody diluent is 0.1% BSA-PBS; SP-2/0 myeloma cell's culture supernatant for negative control hole; Confining liquid is 1% BSA-PBS, every hole dosage 250 μ L; Zymolyte OPD, other is undertaken by standard method;
2. examination criteria toxin sensitivity:
First filter out the optimal combination of envelope antigen concentration and antibody working dilution by chessboard titrimetry, as test condition, the standard of making sterigmatocystin by solid phase antigen indirect competition ELISA method suppresses curve, and result is carried out to Mathematical Statistics Analysis; Wherein ELISA test condition is: the concentration of envelope antigen sterigmatocystin-BSA is 5 μ g/mL, every hole dosage 150 μ L; Antibody working dilution 1:25600; Antibody diluent is 0.1% BSA-PBS; Antibody antigen reactant liquor is every hole 65 μ L antibody+65 μ L respective concentration sterigmatocystins; Confining liquid is 1% BSA-PBS, every hole dosage 250 μ L; Zymolyte is OPD; Sp-2/0 myeloma cell's culture supernatant for negative control, other is undertaken by standard method;
3. the specificity of antibody:
Measure by solid phase antigen indirect competition ELISA method, the toxin of participating in the experiment is sterigmatocystin, aspergillus nidulans, aspergillus flavus, aspergillus parasiticus, aflatoxin horse; Other test condition is same 2.;
4. Subclass of antibody analysis:
Carry out with immune double diffusion method;
5. the affinity of antibody:
Friguet method, the titration of corresponding mouse IgG typical curve goes out IgG antibody content, more corresponding antibody titer curve obtains under equilibrium state, the free antibodies molar concentration [Ab] when antibody makes antigen reach semi-saturation, substitution following formula is obtained the affinity costant K(L/moL of antibody)
Ka＝１/[Ab]
(7) sterigmatocystin extraction and purification in sample
II result and discussion
(1) after the screening Fusion of Cells of Fusion of Cells and hybridoma, grow hybridoma clone in approximately 57. 6% hole, wherein antibody test positive rate is about 88%; Therefrom select the original strong inhibiting hole of antagonism, through subclone repeatedly, set up the hybridoma cell strain of 5 anti-sterigmatocystin monoclonal antibodies of stably excreting, each cell line is after twice subclone, and positive rate has reached 100%; When the 3rd time cloning, there is repeatedly (doubting is false bright property), so carried out subclone 4 times; By above-mentioned antibody purification.
4. according to described in claims Article 1, the standard items of sterigmatocystin are formed by high standard dilution, and dilution is PBS; In 0.1M PBS:1000 mL distilled water, add NaCl 9 g, Na2HPO412H2O 6 g, NaH2PO4 2H2O 0.4 g, pH:7.2.
5. according to described in claims Article 1, the enzyme of the enzyme labelled antibody of sterigmatocystin is by horseradish peroxidase mark, this enzyme labelled antibody is made of cross-linking method, and crosslinking chemical is 25% glutaraldehyde water solution, and principle is to utilize on glutaraldehyde molecule two symmetrical aldehyde radicals, respectively with enzyme and protein molecule in free amino, phenolic group etc. carry out mark with covalent bonds, when mark, should adopt fresh sterling, as the OD of the eleventh of the twelve Earthly Branches dialdehyde as far as possible 235nm/OD 280when nm < 3, the crosslinking chemical being, this invention adopts single stage method to make, and horseradish peroxidase, glutaraldehyde is added and is cross-linked together with aspergillus versicolor antibody, then give excessive glutaraldehyde and prepare the enzyme conjugates with high molecular, due to antibody molecule amount large (16), enzyme molecular weight little (40,000), the amino number of antibody molecule is more much more than zymoprotein molecules of ammonia radix, and antibody-glutaraldehyde that result generates or antibody-glutaraldehyde-antibody are many and cause the activity of enzyme mark thing little, single stage method operation: the 1. preparation of anti-IgG-sterigmatocystin: 10 mg sterigmatocystins are dissolved in to the 1 mLPBS(0.01 moL/L pH7.0 that contains the anti-IgG of 5 mg) in, in ice bath, slowly stir, avoid Bubble formation as far as possible, after dissolving completely, slowly drip 1% glutaraldehyde solution 4 mL, the ultimate density that makes glutaraldehyde is 0.2%, move in room temperature and leave standstill after 2 h ~ 3 h, fully dialyse or remove excessive glutaraldehyde with SephadexG25 post with 4 ℃ of 0.01 moL/L pH 7.0 PBS liquid, 4 ℃ save backup (also can be stored in the mid-low temperature refrigerator of 50% glycerine), 2. the preparation of anti-IgG-HRP: 12 mg HRP are dissolved in containing in 1 mLPBS (0.1 moL/L pH 6.8) of the anti-IgG of 5 mg, add 1% glutaraldehyde solution 4 mL under slowly stirring, put room temperature 2h ~ 3h, fully dialysis or with SephadexG 25except glutaraldehyde, after packing, be stored in low temperature refrigerator in a small amount, avoid multigelation or freeze drying to preserve.
6. according to described in claims, the detecting step of sterigmatocystin is as follows: get coated plate, add standard items/sample 50 μ L in corresponding micropore, add again enzyme labeling thing 50 μ L/ holes, and then add 50 μ L/hole antibody working fluids, vibration mixes gently, reacts 30 min by cover plate membrane cover plate postposition room temperature lucifuge; Carefully open cover plate film, liquid in hole is dried, add cleansing solution 250 μ L/ holes, soak 15 ~ 30 s at every turn, fully wash 4 ~ 5 times, pat dry with thieving paper; Add nitrite ion 100 μ L/holes, vibration mixes gently, with rearmounted 25 ℃ of lucifuge environment reaction 15 min of cover plate membrane cover plate; Every hole adds 50 μ L stop buffers, sets microplate reader at 450 nm places, measures OD value (suggestion 450/630 nm dual wavelength detects, and runs through data in 5 min); The content of ST in calculation sample from typical curve
CN201210435692.3A 2012-11-05 2012-11-05 Preparation and detection method of enzyme-linked immunosorbent assay kit for aspergillus versicolor Pending CN103792360A (en)

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CN111505294A (en) * 2020-04-03 2020-08-07 北京望尔生物技术有限公司 Application of artificial antigen of variolothricin in enzyme linked immunosorbent assay kit

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