CN105399639A - Tyramine artificial antigen and antibody, and preparation methods and application thereof - Google Patents
Tyramine artificial antigen and antibody, and preparation methods and application thereof Download PDFInfo
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- C07C215/46—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
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Abstract
The invention provides a tyramine artificial antigen and antibody, and preparation methods and an application thereof, and relates to preparation of the tyramine artificial antigen and antibody and establishment of an immunoassay method. The problems that a traditional physical and chemical analysis method is tedious and complex, relatively high in cost and slow in analysis speed are overcome, and a simple, rapid, sensitive and accurate immunoassay technique is provided. With adopting of a formaldehyde method and a glutaraldehyde method, tyramine is coupled respectively with cationized bovine serum albumin and ovalbumin, and the tyramine artificial antigen and a tyramine coated antigen are successfully prepared. The artificial antigen is subjected to animal immunization, blood acquirement and separation purification to obtain the specific antibody. A detection method established by using the antibody and used for detection of tyramine in food has good specificity and sensitivity, and the detection limit can be stabilized to 0.02 mg/L; the detection method is low in cost, simple to operate, and suitable for rapid detection of tyramine in the food.
Description
Technical field
The invention belongs to micromolecular compound immunochemistry and trace residue analysis technical field; Relate to organic synthesis, immunochemistry, biological chemistry and materialization measuring technology etc.; In particular to the design and synthesis of alkamines micromolecular compound tyrasamine haptens, artificial antigen, and the preparation of immune animal specific antibody and the foundation of immune analysis method thereof.
Background technology
Tyrasamine is the effective physiologically active substance be present in living organism, is a kind of vasoactive amines, in the eubolism of living organism, plays irreplaceable physiological function.The tyrasamine of trace possesses important regulating effect to pallium and cerebration thereof, and to blood vessel and muscle lax, contractive effect is obvious.In addition, tyrasamine, phenylethylamine etc. have scavenging free radicals, the effect such as anti-oxidant, and can suppress the oxidation of unsaturated fatty acids.Under normal circumstances, human body is taken in a small amount of tyrasamine and can not be had an impact to human body, and when excess intake tyrasamine, histamine (taking in multiple biogenic amine) especially simultaneously time, headache can be produced, feel sick, increased heart rate, elevation of blood pressure, the untoward reaction such as to be short of breath, serious also can life threatening.Tyrasamine also can cause peripheral blood vessel to shrink, and increases the concentration of blood sugar, the secretion of induction norepinephrine, causes patients' blood to raise and migraine etc.EFSA points out, takes in tyrasamine, phenylethylamine, tryptamines etc., can cause vasoconstriction, hypertension after its metabolic exhaustion 30min to a few hours, and with clinical symptom such as headache, perspiration, Nausea and vomiting, pupil dilations.Under normal circumstances, can recover gradually at 24h.Research shows that the food being rich in tyrasamine can bring out migraine, if the content of tyrasamine of ingesting can cause elevation of blood pressure and migraine more than 100mg, if can cause acute toxic swelling more than 1080mg/kg.
Tyrasamine is extensively present in all kinds of leavened food such as meat and its products, fishery products and beer, soy sauce, cheese, its contents level is usually as the deliberated index of freshness and sanitary condition, and therefore a lot of method for separating and detecting of Chinese scholars to tyrasamine is studied.The method of common detection amine substance has chromatography, capillary electrophoresis and hexavalent chrome bio-removal etc.There is complex pretreatment in traditional instrument method, instrument and corresponding support expense costliness, and the shortcomings such as analysis speed is slow, are difficult to the needs meeting actual analysis, the analytical technology that therefore an urgent demand development is easy, quick, sensitive.
But different from macromole, micromolecular compound immunoassay has own characteristic:
(1) micromolecular compound (MW≤1000dolton) does not generally have artificial antigen property, specific antibody can not be produced, the haptens of outstanding molecule stereo structure specific site must be synthesized by direct immunization animal, and connecting and composing joiner with macromolecular carrier, ability immune animal produces the specific antibody for this target micromolecular compound.The binding substances of this haptens and macromolecular carrier is called artificial antigen.The preparation of artificial antigen is not arbitrary, comprise binding site, combination, kind of carrier and haptens and any structural difference of target analytes as the factors of size, shape, composition, configuration, conformation, polarity, cloud density etc., all greatly may affect the character of corresponding antibodies, therefore they are the keys determining to produce its specific antibody and set up immune analysis method.
(2) although micromolecular compound does not have artificial antigen property, there is reactionogenicity, namely there is the ability with corresponding antibodies generation immunological response, and can Quantitative in vitro be carried out, follow the law of mass action.
Immuno analytical method is introduced into small molecules residual point of folding field, become a kind of quantitative analysis tech having development and application potential most it-, paid attention to widely.The key of this technical study is the preparation of haptenic molecular designing, synthesis and artificial holoantigen and antibody.Therefore, target analyte molecule immunological characteristic, and how by chemistry or biochemical technology outstanding and utilize these characteristics, be the important research contents in this field.This technology has become a brand-new field of trace analysis research at present, can with traditional analysis side by side as a new analysis approach.Tyrasamine artificial antigen and specific antibody and set up immune analysis method based on this there is not been reported.
Summary of the invention
The object of the invention is by design and synthesis tyrasamine haptens and corresponding artificial antigen and antibody.Another object of the present invention is to provide the haptens of tyrasamine and the preparation method of corresponding artificial antigen and antibody.Further object of the present invention is to provide the haptens of tyrasamine and the application of corresponding artificial antigen and antibody.Wherein the artificial antigen of tyrasamine comprises artificial antigen and coating antigen.
Unique distinction of the present invention is to highlight tyrasamine molecular specificity antigenic determinant, overcomes again the difficulty of chemosynthesis, and immune animal induction produces the very high specific antibody of affinity; And establish ELISA method and rapid immunoassay method based on this, accurately detect the tyramine content in food.
The present invention utilizes target analytes haptens and carrier protein coupling, prepare effective artificial antigen, immune animal preparation is to small molecule analysis thing specific antibody, the specificity immunology of antigen-antibody is utilized to react, thus qualitative, quantitative ground detects ultramicron small molecules target analytes in sample, namely can be used for sample and measure.Its selectivity is decided by the specificity of immunological response, and the affinity of antibody and the property examined of marker are depended in its sensitivity.Therefore can analyzing and testing tyrasamine content in the sample rapidly and accurately.The key of this technical study is the preparation of haptenic molecular designing, synthesis and artificial holoantigen and antibody.
Artificial antigen is the conjugate of synthesis haptens and carrier proteins, if for immune animal, is artificial antigen, if for wrapping by Sptting plate in immunoassay, be coating antigen.For same target molecule, artificial antigen and coating antigen are usually without same conjugate, and object reduces antibody to the non-specific binding of coating antigen, improves the specificity and sensitivity analyzed.Because the antibody that artificial antigen immune animal produces, can not only haptens be identified, also can identification carrier albumen.Therefore, the artificial antigen carrier proteins used with coating antigen is generally not identical, and structure and the molecular mass difference of carrier proteins are larger, and non-specific binding reaction is more weak, and sensitivity for analysis is higher.In artificial antigen and coating antigen, haptens structure is variant in addition is also conducive to the sensitivity of raising method.The present invention adopts diverse ways in order that obtain the sensitivity of raising method in the preparation of artificial antigen and coating antigen.
For achieving the above object, technical scheme of the present invention is achieved in that
The haptenic molecular structure of a kind of tyrasamine is as follows:
A kind of tyrasamine artificial antigen, be to be connected with the bovine serum albumin of cationization by tyrasamine haptens to synthesize, described tyrasamine artificial antigen molecular structural formula is:
A kind of tyrasamine coating antigen, be to be connected with ovalbumin by tyrasamine haptens to synthesize, described tyrasamine coating antigen molecular structural formula is:
A kind of tyrasamine antibody, be can with the IgG antibody of artificial antigen or coating antigen generation specific immune response.
The preparation method of tyrasamine artificial antigen adopts formaldehyde method fully to expose all antigenic determinants of tyrasamine, comprises the following steps:
(1) preparation of cationization BSA (cBSA):
Accurately measure 20mLPBS, under ice bath, slowly add 18.0mg quadrol (EDA) while stirring, adjust pH to 7.4 with 1mol/LHCl; Taking 1.00gBSA and 56.0mgEDC adds in above-mentioned solution, room temperature reaction 2h; With glass hourglass bucket suction filtration, get supernatant liquor and load in dialysis tubing; Dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then at-20 DEG C, carry out drying with vacuum refrigerating machine, finally frozen in-20 DEG C of refrigerators.
(2) connection of tyrasamine and carrier proteins cBSA:
Accurately take 20.0mgcBSA in 25mL round-bottomed flask, the PBS adding 4mL0.01mol/L is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved.Tyrasamine is slowly added in the albumen of dissolving.Add the formalin of 5 μ L37%, 30 DEG C of water-bath magnetic agitation 8h.Reaction solution is clarified, and loads in dialysis tubing, and dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
The preparation method of tyrasamine coating antigen adopts glutaraldehyde one step connection method, comprises the following steps:
Accurately take 20.0mgOVA in 25mL round-bottomed flask, the PBS adding 4mL0.01M is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved.Tyrasamine is slowly added in the OVA of dissolving.Slowly add the glutaraldehyde water solution of 15 μ L25%, 4 DEG C of lower magnetic forces stir and spend the night.Loaded by reaction solution in dialysis tubing, dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
The preparation method of tyrasamine antibody, comprises the following steps:
(1) immunity: immune animal selects female White Rabbit, first immunisation adopts back multiple spot subcutaneous injection and leg muscle injecting immune, and booster immunization adopts back multiple spot subcutaneous injection immunity.Carry out four booster immunizations after just exempting from, specific practice respectively:
Initial immunity: get in the NaCl solution and the solution prepared of Freund's complete adjuvant equal-volume that the above-mentioned artificial antigen of 1mg is dissolved in 0.9%, carry out animal immune;
Booster immunization: be dissolved in NaCl solution and the solution prepared of Freund's incomplete adjuvant equal-volume of 0.9% with the above-mentioned artificial antigen of 0.5mg, carry out animal immune; Booster immunization respectively at immunity behind 2 weeks, 4 weeks and 6 weeks after initial immunity three times, after this one month, interval.Get blood by the auricular vein of rabbit during latter 9 days of the 5th immunity, carry out bioactivity;
(2) antibody purification: periodic monitor animal antibody titer, when antibody to the above-mentioned tyrasamine coating antigen stated tire reach higher level time, gather blood, and centrifugal acquisition antiserum(antisera), use ProteinA-Sepharose4B albumen affinity column antagonistic Serum to carry out purifying, prepare above-mentioned IgG antibody.
Described tyrasamine antibody is applied to the immunodetection of biogenic amine tyramine content, and described immunodetection is enzyme-linked immunosorbent assay, i.e. ELISA.
Tyrasamine antibody is applied to the immunodetection of biogenic amine tyramine content, comprises the steps:
(1) bag quilt: envelope antigen is suitably diluted with coating buffer, is then coated on 96 hole enzyme plates with 100 μ L/well, and 4 DEG C of overnight incubation or 37 DEG C hatch 3h.
(2) wash plate: add washings PBST, 250 μ L/ holes, vibrate 2min on vibrator, discards PBST, repeats to wash plate 3 times;
(3) close: every hole adds 200 μ L confining liquids, in 37 DEG C of closed 1h, then discards confining liquid, repeats to wash plate 3 times with PBST;
(4) application of sample: every hole first adds tyrasamine standardized solution or the sample solution of 50 μ L, and then respectively add the antibody of 50 μ L, after application of sample, competing reaction 1h at 37 DEG C, then repeats to wash plate 4 times with PBST;
(5) add goat-anti rabbit HRP mark two to resist: goat-anti rabbit HRP is marked two anti-PBS and dilutes proper concn, 100 μ L/well application of samples, hatch 30min at 37 DEG C, repeat to wash plate 5 times with PBST.
(6) develop the color: every hole adds the substrate solution after 100 μ L mixings, reacts 15-20min at 37 DEG C;
(7) stop: every hole adds the sulfuric acid stop buffer of 50 μ L, color development stopping is reacted.
(8) reading: the absorbance measuring each hole under dual wavelength mode (for measuring wavelength, 650nm is reference wavelength to 450nm) by microplate reader.The calculation formula of inhibiting rate is such as formula shown:
In formula: OD contrasts: the light absorption value adding the micropore of PBS and antibody;
OD is blank: the light absorption value only adding the micropore of PBS;
The present invention prepares the highly sensitive of tyrasamine, the antibody of high specificity, sets up simply, tyraminase linked immune detection method fast, and the detection for food freshness provides a kind of technical support reliably.
The present invention has following advantage and effect relative to prior art:
(1) artificial antigen of this design, synthesis and target determinand similarity degree high, retain complete to the feature structure of determinand, fully expose tyrasamine antigenic determinant, the antibody good for preparation specificity is laid a good foundation; Adopt different synthetic method to prepare the coating antigen having textural difference, form antigen-antibody competing reaction system with antibody, utilize the sensitivity of antibody recognition differentiation raising method.
(2) its antibody has good specificity and sensitivity, and limit of detection reaches 0.02mg/L.
(3) therefore, the present invention synthesizes the method for artificial antigen compared with additive method, is more easy to popularize.And the method for quick provided is easy and simple to handle, quick, and the tolerance range detected can reach more than 90%, is applicable to very much the needs of Site Detection.Therefore, the present invention not only does well in test in laboratory, and for developing enzyme linked immunological fast detecting tool with low cost, that detection efficiency is high, easy and simple to handle, laying a good foundation, having a good application prospect; Not only have an economic benefit but also have social benefit.
Accompanying drawing explanation
Fig. 1 is tyrasamine enzyme linked immunosorbent detection typical curve
Embodiment
Embodiment one
(1) preparation of tyrasamine artificial antigen
A. the preparation of cationization BSA (cBSA):
Accurately measure 20mLPBS, under ice bath, slowly add 18.0mg quadrol (EDA) while stirring, adjust pH to 7.4 with 1mol/LHCl; Taking 1.00gBSA and 56.0mgEDC adds in above-mentioned solution, room temperature reaction 2h; With glass hourglass bucket suction filtration, get supernatant liquor and load in dialysis tubing; Dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then at-20 DEG C, carry out drying with vacuum refrigerating machine, finally frozen in-20 DEG C of refrigerators.
B. the connection of tyrasamine and carrier proteins cBSA:
Accurately take 20.0mgcBSA in 25mL round-bottomed flask, the PBS adding 4mL0.01mol/L is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved.Tyrasamine is slowly added in the albumen of dissolving.Add the formalin of 5 μ L37%, 30 DEG C of water-bath magnetic agitation 8h.Reaction solution is clarified, and loads in dialysis tubing, and dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
(2) preparation of tyrasamine coating antigen
Accurately take 20.0mgOVA in 25mL round-bottomed flask, the PBS adding 4mL0.01M is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved.Tyrasamine is slowly added in the OVA of dissolving.Slowly add the glutaraldehyde water solution of 15 μ L25%, 4 DEG C of lower magnetic forces stir and spend the night.Loaded by reaction solution in dialysis tubing, dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
(3) preparation of tyrasamine antibody
A. immunity: immune animal selects female White Rabbit, first immunisation adopts back multiple spot subcutaneous injection and leg muscle injecting immune, and booster immunization adopts back multiple spot subcutaneous injection immunity.Carry out four booster immunizations after just exempting from, specific practice respectively:
Initial immunity: get in the NaCl solution and the solution prepared of Freund's complete adjuvant equal-volume that the above-mentioned artificial antigen of 1mg is dissolved in 0.9%, carry out animal immune;
Booster immunization: be dissolved in NaCl solution and the solution prepared of Freund's incomplete adjuvant equal-volume of 0.9% with the above-mentioned artificial antigen of 0.5mg, carry out animal immune; Booster immunization respectively at immunity behind 2 weeks, 4 weeks and 6 weeks after initial immunity three times, after this one month, interval.Get blood by the auricular vein of rabbit during latter 9 days of the 5th immunity, carry out bioactivity;
B. antibody purification: periodic monitor animal antibody titer, when antibody to tyrasamine coating antigen according to claim 3 tire reach higher level time, gather blood, and centrifugal acquisition antiserum(antisera), use ProteinA-Sepharose4B albumen affinity column antagonistic Serum to carry out purifying, prepare IgG antibody according to claim 4.
Utilize square formation to test and determine that the package amount of coating antigen is every hole 0.1 μ g respectively, antibody dilution multiple is 1:1000, ELIAS secondary antibody extension rate is 1:15000, measures antibodies specific with this understanding.With the cross reaction degree of antibody and structurally similar compounds, with suppress antibody Bmax 50% needed for the concentration IC of target analytes
50with the concentration IC of required various structurally similar compounds
50the percentage ratio of ratio represent, i.e. cross reacting rate CR (%).
CR (%)=IC
50(tyrasamine)/IC
50(analog) × 100 cross reaction is less, and antibodies specific is higher.In table 1.
Table 1 tyrasamine antibody is to tyrasamine and analog cross reacting rate thereof
Embodiment 2
The tyrasamine antibody using embodiment 1 to obtain carries out immunodetection
(1) bag quilt: envelope antigen is suitably diluted with coating buffer, is then coated on 96 hole enzyme plates with 100 μ L/well, and 4 DEG C of overnight incubation or 37 DEG C hatch 3h.
(2) wash plate: add washings PBST, 250 μ L/well, vibrate 2min on vibrator, discards PBST, repeats to wash plate 3 times;
(3) close: every hole adds 200 μ L confining liquids, in 37 DEG C of closed 1h, then discards confining liquid, repeats to wash plate 3 times with PBST;
(4) application of sample: every hole first adds tyrasamine standardized solution or the sample solution of 50 μ L, and then respectively add the antibody of 50 μ L, after application of sample, competing reaction 1h at 37 DEG C, then repeats to wash plate 4 times with PBST;
(5) add goat-anti rabbit HRP mark two to resist: goat-anti rabbit HRP is marked two anti-PBS and dilutes proper concn, 100 μ L/well application of samples, hatch 30min at 37 DEG C, repeat to wash plate 5 times with PBST.
(6) develop the color: every hole adds the substrate solution after 100 μ L mixings, reacts 15-20min at 37 DEG C;
(7) stop: every hole adds the sulfuric acid stop buffer of 50 μ L, color development stopping is reacted.
(8) reading: the absorbance measuring each hole under dual wavelength mode (for measuring wavelength, 650nm is reference wavelength to 450nm) by microplate reader.The calculation formula of inhibiting rate is such as formula shown:
In formula: OD contrasts: the light absorption value adding the micropore of PBS and antibody;
OD is blank: the light absorption value only adding the micropore of PBS;
In typical curve, X-axis is tyrasamine concentration, and Y-axis is the inhibiting rate of tyrasamine standard substance antagonist at various concentrations, is typical S type curve.
As shown in Figure 1, the sensitivity (IC of tyrasamine indirect competitive ELISA method is calculated
50value) and detection limit (IC
15value) be respectively: 0.20 ± 0.015mg/L and 0.02 ± 0.004mg/L.
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (9)
1. a tyrasamine haptens, is characterized in that, molecular structural formula is:
2. a tyrasamine artificial antigen, is characterized in that, to be connected with cationization bovine serum albumin by tyrasamine haptens according to claim 1 and to synthesize, described tyrasamine artificial antigen molecular structural formula is:
3. a tyrasamine coating antigen, is characterized in that, to be connected with ovalbumin by tyrasamine haptens according to claim 1 and to synthesize, the tyrasamine coating antigen molecular structural formula obtained is:
4. a tyrasamine antibody, is characterized in that, the IgG antibody of described tyrasamine antibody capable and claim 2 artificial antigen or claim 3 coating antigen generation specific immune response.
5. the preparation method of tyrasamine artificial antigen according to claim 2, is characterized in that, comprise the following steps:
(1) preparation of cationization BSA:
Accurately measure 20mLPBS, under condition of ice bath, slowly add 18.0mg quadrol while stirring, adjust pH to 7.4 with 1mol/LHCl; Taking 1.00gBSA and 56.0mgEDC adds in above-mentioned solution, room temperature reaction 2h; With glass hourglass bucket suction filtration, get supernatant liquor and load in dialysis tubing; Dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then at-20 DEG C, carry out drying with vacuum refrigerating machine, finally frozen in-20 DEG C of refrigerators;
(2) connection of tyrasamine and carrier proteins cBSA:
Accurately take 20.0mgcBSA in 25mL round-bottomed flask, the PBS adding 4mL0.01mol/L is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved; Tyrasamine is slowly added in the albumen of dissolving; Add the formalin of 5 μ L37%, 30 DEG C of water-bath magnetic agitation 8h, reaction solution is clarified, and loads in dialysis tubing, and dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
6. the preparation method of tyrasamine coating antigen according to claim 3, is characterized in that, comprise the following steps:
Accurately take 20.0mgOVA in 25mL round-bottomed flask, the PBS adding 4mL0.01M is dissolved, and accurately takes 4.2mg tyrasamine in the little brown bottle of 5mL, adds 200 μ LDMSO and dissolved; Tyrasamine is slowly added in the OVA of dissolving; Slowly add the glutaraldehyde water solution of 15 μ L25%, 4 DEG C of lower magnetic forces stir and spend the night; Loaded by reaction solution in dialysis tubing, dialyse 3 days continuously in 4 DEG C with the PBS of 0.01mol/L, then packing ,-20 DEG C save backup.
7. the preparation method of tyrasamine antibody described in claim 4, is characterized in that, comprise the following steps:
Immune animal selects female White Rabbit, and first immunisation adopts back multiple spot subcutaneous injection and leg muscle injecting immune, and booster immunization adopts back multiple spot subcutaneous injection immunity; Carry out four booster immunizations after just exempting from, specific practice respectively:
Initial immunity: get in the NaCl solution and the solution prepared of Freund's complete adjuvant equal-volume that the above-mentioned artificial antigen of 1mg is dissolved in 0.9%, carry out animal immune;
Booster immunization: be dissolved in NaCl solution and the solution prepared of Freund's incomplete adjuvant equal-volume of 0.9% with the above-mentioned artificial antigen of 0.5mg, carry out animal immune; Booster immunization respectively at immunity behind 2 weeks, 4 weeks and 6 weeks after initial immunity three times, after this one month, interval; Get blood by the auricular vein of rabbit during latter 9 days of the 5th immunity, carry out bioactivity;
Antibody purification: periodic monitor animal antibody titer, when antibody to tyrasamine coating antigen according to claim 3 tire reach higher level time, gather blood, and centrifugal acquisition antiserum(antisera), use ProteinA-Sepharose4B albumen affinity column antagonistic Serum to carry out purifying, prepare IgG antibody according to claim 4.
8. the application of tyrasamine antibody described in claim 4, is characterized in that, described tyrasamine antibody is applied to the immunodetection of tyramine content.
9. the application of tyrasamine antibody according to claim 8, is characterized in that comprising the steps:
(1) bag quilt: envelope antigen is diluted with coating buffer, is then coated on 96 hole enzyme plates with 100 μ L/well, and 4 DEG C of overnight incubation or 37 DEG C hatch 3h;
(2) wash plate: add washings PBST, 250 μ L/ holes, vibrate 2min on vibrator, discards PBST, repeats to wash plate 3 times;
(3) close: every hole adds 200 μ L confining liquids, in 37 DEG C of closed 1h, then discards confining liquid, repeats to wash plate 3 times with PBST;
(4) application of sample: every hole first adds tyrasamine standardized solution or the sample solution of 50 μ L, and then respectively add the antibody of 50 μ L, after application of sample, competing reaction 1h at 37 DEG C, then repeats to wash plate 4 times with PBST;
(5) add goat-anti rabbit HRP mark two to resist: goat-anti rabbit HRP is marked two anti-PBS and dilutes proper concn, 100 μ L/well application of samples, hatch 30min at 37 DEG C, repeat to wash plate 5 times with PBST;
(6) develop the color: every hole adds the substrate solution after 100 μ L mixings, reacts 15-20min at 37 DEG C;
(7) stop: every hole adds the sulfuric acid stop buffer of 50 μ L, color development stopping is reacted;
(8) reading: in dual wavelength mode, 450nm is for measuring wavelength, and 650nm is reference wavelength, and lower microplate reader measures the absorbance in each hole.The calculation formula of inhibiting rate is such as formula shown:
In formula: OD contrasts: the light absorption value adding the micropore of PBS and antibody;
OD is blank: the light absorption value only adding the micropore of PBS;
OD: the absorbance having added the micropore of antibody and standard substance or sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239387A (en) * | 2020-01-18 | 2020-06-05 | 天津科技大学 | Fluorescence immunoassay method for simultaneously detecting tyramine and histamine |
| CN111499637A (en) * | 2020-04-15 | 2020-08-07 | 华南农业大学 | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof |
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| CN114252591A (en) * | 2021-12-27 | 2022-03-29 | 深圳市亚辉龙生物科技股份有限公司 | Magnetic bead coating and preparation method thereof and detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597233A (en) * | 2008-04-15 | 2009-12-09 | 曾立波 | Not with the methyl amphetamine monoclonal antibody kit of ephedrine and pseudoephedrine cross reaction |
| US20110207171A1 (en) * | 2006-02-10 | 2011-08-25 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
| CN104672322A (en) * | 2014-12-05 | 2015-06-03 | 上海烟草集团有限责任公司 | Phenol artificial antigen synthetic method |
-
2015
- 2015-12-14 CN CN201510937173.0A patent/CN105399639A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110207171A1 (en) * | 2006-02-10 | 2011-08-25 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
| US20140377837A1 (en) * | 2006-02-10 | 2014-12-25 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
| CN101597233A (en) * | 2008-04-15 | 2009-12-09 | 曾立波 | Not with the methyl amphetamine monoclonal antibody kit of ephedrine and pseudoephedrine cross reaction |
| CN104672322A (en) * | 2014-12-05 | 2015-06-03 | 上海烟草集团有限责任公司 | Phenol artificial antigen synthetic method |
Non-Patent Citations (1)
| Title |
|---|
| 孙丛丛: "酪胺酶联免疫检测方法的研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239387A (en) * | 2020-01-18 | 2020-06-05 | 天津科技大学 | Fluorescence immunoassay method for simultaneously detecting tyramine and histamine |
| CN111239387B (en) * | 2020-01-18 | 2023-06-23 | 天津科技大学 | Fluorescent immunization method for simultaneously detecting tyramine and histamine |
| CN111499637A (en) * | 2020-04-15 | 2020-08-07 | 华南农业大学 | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof |
| CN111499637B (en) * | 2020-04-15 | 2021-06-15 | 华南农业大学 | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof |
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| CN112028786B (en) * | 2020-08-12 | 2022-02-11 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| CN114252591A (en) * | 2021-12-27 | 2022-03-29 | 深圳市亚辉龙生物科技股份有限公司 | Magnetic bead coating and preparation method thereof and detection kit |
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