CN101885768B - Mercury ion antigens and preparation method and application thereof - Google Patents
Mercury ion antigens and preparation method and application thereof Download PDFInfo
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- CN101885768B CN101885768B CN200910083947A CN200910083947A CN101885768B CN 101885768 B CN101885768 B CN 101885768B CN 200910083947 A CN200910083947 A CN 200910083947A CN 200910083947 A CN200910083947 A CN 200910083947A CN 101885768 B CN101885768 B CN 101885768B
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Abstract
The invention discloses mercury ion antigens and a preparation method and application thereof. The preparation method for the mercury ion antigens comprises the following steps of: (1) reacting acid solution of para-aminobenzylethylene diamine tetraacetic acid with soluble nitrite to obtain diazotized para-aminobenzylethylene diamine tetraacetic acid; (2) reacting the diazotized para-aminobenzylethylene diamine tetraacetic acid in the step (1) with carrier protein to obtain a coupling product; and (3) reacting the coupling product in the step (2) with mercury ions to obtain the mercury ion antigens. The preparation method for the mercury ion antigens can conveniently and rapidly prepare the mercury ion antigens, and has the advantages of compact synthesis steps, low synthesis cost and good effect. Antibodies obtained by immunization of the mercury ion antigens prepared by the method have good specificity and low minimum detection limit. The method for preparing the mercury ion antigens and the mercury ion antigens obtained by the method have broad application prospect in enzyme-linked immunological detection of mercury ions.
Description
Technical field
The present invention relates to a kind of mercury ion antigen and preparation method thereof and application.
Background technology
Mercury is the important pollution substance in environment and the agricultural-food, owing to it can be accumulated in human body and environment for a long time, and can pass to the human or animal through the inrichment of food chain, has therefore brought serious harm to human health.Like the mid-50 in Japan once because of world-shaking minamata disease has appearred in the fish that edible heavy metal Hg pollutes, thereby make the harm problem of mercury pollution in the environment obtain global concern.Accumulating in a large number of mercury mainly is unordered, the discharging of transfiniting because of three industrial wastes in the environment, thereby causes a large amount of soil and water pollution, causes environmental degradation.
At present, the analytical procedure of heavy metal Hg mainly contains atomic absorption spectrometry, inductively coupled plasma emission light/mass spectroscopy, x-ray fluorescence spectrometry method and potentiometric stripping analysis method etc.These several kinds of detection methods detect heavy metal Hg not only needs expensive plant and instrument, and testing cost is high, detection time is long and it is numerous and diverse to detect step, can not be used for on-the-spot rapid detection.In addition; The toxicity of heavy metal is relevant with its chemical species, physical condition and the state of oxidation; And above-mentioned instrumental method can't obtain the state of oxidation or the differentiation information of heavy metal mostly, can only obtain the total amount of a certain heavy metal element, and this macroanalysis is not directly proportional with toxicity sometimes.Compare with instrumental method, that immunoassay has is quick, easy, real-time, the scene that is easy to carry out is detected, sample preparation is simple, highly sensitive, selectivity strong, be suitable for advantage such as high throughput analysis, and can also reduce the detection cost significantly.With immunoassay can a certain predetermined substance of rapid detection content, but to be applied to the detection metal mercury ions to immune analysis method, also must preparation corresponding antigen and antibody.
Summary of the invention
The purpose of this invention is to provide a kind of mercury ion antigen and preparation method thereof.
The preparation method of mercury ion antigen provided by the present invention may further comprise the steps:
(1) with the acid solution and the reaction of solubility nitrite of p-aminophenyl methylethylenediaminetetraacetic acid, obtains diazotizing p-aminophenyl methylethylenediaminetetraacetic acid;
(2) with the diazotizing p-aminophenyl methylethylenediaminetetraacetic acid and the carrier proteins reaction of above-mentioned steps (1), obtain coupled product;
(3) with the coupled product and the mercury ion reaction of above-mentioned steps (2), obtain mercury ion antigen.
In the aforesaid method, in the said step (1), the acid solution of said p-aminophenyl methylethylenediaminetetraacetic acid is that the p-aminophenyl methylethylenediaminetetraacetic acid is dissolved in hydrochloric acid, sulfuric acid, nitric acid, crosses the acid solution that obtains in chloric acid or the fluoroboric acid; Hydrionic mol ratio is 1 in said p-aminophenyl methylethylenediaminetetraacetic acid and the said acid solution: (4-10), specifically can be 1: 6.
In the said step (1), the mol ratio of said p-aminophenyl methylethylenediaminetetraacetic acid and said solubility nitrite is 1: (1-3), specifically can be 1: 2.
In the aforesaid method, in the said step (2), said carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or oralbumin; The mol ratio of said carrier proteins and said diazotizing p-aminophenyl methylethylenediaminetetraacetic acid is 1: (5-25), specifically can be 1: 10.
In the aforesaid method, in the said step (3), the mol ratio of said conjugate and said mercury ion is 1: (1-10), specifically can be 1: 5.
In the aforesaid method, the reaction conditions of said step (1) is: 0-5 ℃ of reaction under the lucifuge condition; Specifically can be the following 4 ℃ of reactions of lucifuge condition.
In the aforesaid method, the reaction conditions of said step (2) and step (3) is: temperature of reaction is 15-25 ℃, and the pH value is 8.5-10.5, and the reaction times is 0.5-24h; Specifically can be: temperature of reaction is 25 ℃, and the pH value is 9.5, and the reaction times is 12h.
In the aforesaid method, in the said step (2), before said carrier proteins and the said diazotizing p-aminophenyl methylethylenediaminetetraacetic acid reaction, earlier said carrier proteins is dissolved in the carbonate buffer solution; The pH of said carbonate buffer solution is 9.0-9.6, specifically can be 9.5.
In the aforesaid method, in the said step (2), also comprise the step that said conjugate is dialysed;
In the said step (3), also comprise the step that said mercury ion antigen is dialysed, to remove mercury ion and other small-molecule substance that is not closed by huge legendary turtle.
The mercury ion antigen that adopts above-mentioned arbitrary method to obtain also belongs to protection scope of the present invention.
Above-mentioned mercury ion antigen can be used for environment, water body, food or mercury in soils ionic and detects.
The method for preparing mercury ion antigen of the present invention can obtain mercury ion antigen quickly and easily, and synthesis step is short and sweet, synthetic cost is low, effective.Carry out with the mercury ion antigen of the inventive method preparation that the specificity of the antibody that immunity obtains is good, the lowest detection limit value is low.The mercury ion antigen for preparing the method for mercury ion antigen and obtained by this method of the present invention will have broad application prospects in the enzyme linked immunosorbent detection of mercury ion.
Description of drawings
Fig. 1 is the synthetic route chart of mercury ion antigen.
Fig. 2 is for EDTA-Hg being the mercury ion indirect elisa method typical curve that standard model is set up.
Embodiment
Experimental technique described in the following embodiment like no specified otherwise, is ordinary method; Said reagent and biomaterial like no specified otherwise, all can obtain from commercial sources.
P-aminophenyl methylethylenediaminetetraacetic acid (available from Sigma company, cat. no is A3473), goat anti-mouse igg-HRP is (available from Jackson company; Cat. no is 79556), Freund's complete adjuvant (available from Sigma company, cat. no is F5881); Freund's incomplete adjuvant (available from Sigma company, cat. no is F5506), EDTA is (available from Sigma company; Cat. no is E6758), bovine serum albumin (available from Sigma company, cat. no is A7906); Oralbumin (available from Sigma company, cat. no is A5503), HgCl
2(available from Sigma company, cat. no is 449199), all the other HCl, K
2CO
3, NaNO
2With conventional reagent such as glycerine all available from Beijing chemical reagents corporation.
Embodiment 1, the antigenic preparation of mercury-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin (Hg-p-aminophenyl methyl EDTA-BSA)
The synthetic route chart of mercury ion antigen is as shown in Figure 1.
1, the diazotization of p-aminophenyl methylethylenediaminetetraacetic acid
(1) takes by weighing the solid p-aminophenyl methylethylenediaminetetraacetic acid of 3.0mg, fully be dissolved among the HCl of 0.5mL 0.1M, make that hydrionic mol ratio is 1: 6 among p-aminophenyl methylethylenediaminetetraacetic acid and the HCl;
(2) solution with above-mentioned steps (1) carries out magnetic agitation under 4 ℃ of lucifuge conditions, and every interval 30s drips 30 μ L quality percentage compositions in solution be 1% NaNO
2Solution; NaNO
2Add-on be excessive, i.e. NaNO
2With the mol ratio of p-aminophenyl methylethylenediaminetetraacetic acid more than or equal to 2: 1;
(3) with the starch-kalium iodide test paper reaction solution of above-mentioned steps (2) is tested, after test paper becomes basket, continue reaction 15 minutes, obtain diazotization p-aminophenyl methylethylenediaminetetraacetic acid solution.
2, diazotization p-aminophenyl methylethylenediaminetetraacetic acid and carrier proteins carry out coupling
(1) takes by weighing 50.0mg bovine serum albumin (BSA), fully be dissolved in the carbonate buffer solution of 2.0mL pH 9.6;
(2) the diazotization p-aminophenyl methylethylenediaminetetraacetic acid solution that above-mentioned steps 1 is obtained dropwise adds in the BSA solution of above-mentioned steps (1), and the mol ratio that makes p-aminophenyl methylethylenediaminetetraacetic acid and BSA is 10: 1;
Under (3) 25 ℃ of conditions, with the reaction solution stirred overnight (12 hours) of above-mentioned steps (2), during to use the quality percentage composition be 5% K
2CO
3The pH of reaction solution is controlled at 9.5;
(4) reaction placed dialysis tubing with the reaction solution of above-mentioned steps (3) after 12 hours, and using pH is 9.5 PBS dialysis 2 days, changes dialyzate every day 3 times, obtains the diazotization p-aminophenyl methylethylenediaminetetraacetic acid of purifying and the conjugate of carrier proteins.
3, the coupling of heavy metal ion mercury
(1) takes by weighing 10.0mg HgCl
2, be mixed with 0.1M HgCl with ultrapure water
2Solution;
(2) with the HgCl of above-mentioned steps (1)
2Solution dropwise joins in the conjugate solution of above-mentioned steps 2 acquisitions, stirs while dripping, and the mol ratio that makes diazotization p-aminophenyl methylethylenediaminetetraacetic acid and mercury ion is 1: 5;
(3) solution with above-mentioned steps (2) stirred 24 hours under 25 ℃ of conditions, and using pH is 9.5 PBS dialysis 2 days, changes dialyzate every day 3 times, with remove the mercury that do not connect from and other impurity, the dialysis product is Hg-p-aminophenyl methyl EDTA-BSA antigenic solution;
(4) with the dialysis product packing of above-mentioned steps (3), through-40 ℃ freezing after, vacuum-drying, it is subsequent use to place-20 ℃ of refrigerators to preserve.
1, the diazotization of p-aminophenyl methylethylenediaminetetraacetic acid
(1) take by weighing the solid p-aminophenyl methylethylenediaminetetraacetic acid of 3.0mg, fully be dissolved among the HCl of 0.5mL 0.1M, the mol ratio that makes p-aminophenyl methylethylenediaminetetraacetic acid and HCl is 1: 6;
(2) solution with above-mentioned steps (1) carries out magnetic agitation under 4 ℃ of lucifuge conditions, and every interval 30s drips 30 μ L quality percentage compositions in solution be 1% NaNO
2Solution; NaNO
2Add-on be excessive, i.e. NaNO
2With the mol ratio of p-aminophenyl methylethylenediaminetetraacetic acid more than or equal to 2: 1;
(3) with the starch-kalium iodide test paper reaction solution of above-mentioned steps (2) is tested, after test paper becomes basket, continue reaction 15 minutes, obtain diazotization p-aminophenyl methylethylenediaminetetraacetic acid solution.
2, diazotization p-aminophenyl methylethylenediaminetetraacetic acid and carrier proteins carry out coupling
(1) takes by weighing 34.0mg oralbumin (OVA), fully be dissolved in the carbonate buffer solution of 2.0mL pH 9.6;
(2) the diazotization p-aminophenyl methylethylenediaminetetraacetic acid solution that above-mentioned steps 1 is obtained dropwise adds in the OVA solution of above-mentioned steps (1), and the mol ratio that makes p-aminophenyl methylethylenediaminetetraacetic acid and OVA is 10: 1;
Under (3) 25 ℃ of conditions, with the reaction solution stirred overnight (12 hours) of above-mentioned steps (2), during to use the quality percentage composition be 5% K
2CO
3The pH of reaction solution is controlled at 9.5;
(4) reaction placed dialysis tubing with the reaction solution of above-mentioned steps (3) after 12 hours, and using pH is 9.5 PBS dialysis 2 days, changes dialyzate every day 3 times, obtains the diazotization p-aminophenyl methylethylenediaminetetraacetic acid of purifying and the conjugate of carrier proteins.
3, the coupling of heavy metal ion mercury
(1) takes by weighing 10.0mg HgCl
2, be mixed with 0.1M HgCl with ultrapure water
2Solution;
(2) with the HgCl of above-mentioned steps (1)
2Solution dropwise joins in the conjugate solution of above-mentioned steps 2 acquisitions, stirs while dripping, and the mol ratio that makes diazotization p-aminophenyl methylethylenediaminetetraacetic acid and mercury ion is 1: 5;
(3) solution with above-mentioned steps (2) stirred 24 hours under 25 ℃ of conditions, and using pH is 9.5 PBS dialysis 2 days, changes dialyzate every day 3 times, with remove the mercury that do not connect from and other impurity, the dialysis product is Hg-p-aminophenyl methyl EDTA-OVA antigenic solution;
(4) with the dialysis product packing of above-mentioned steps (3), through-40 ℃ freezing after, vacuum-drying places-20 ℃ of refrigerators to preserve.
(5) get the lyophilized products of 2mg above-mentioned steps (4), use by PBS and glycerine (volume ratio of PBS and glycerine is 1: the 1) mixed solution of forming and be diluted to the concentration of 1mg/mL, be stored in-20 ℃ of refrigerators.
The application of embodiment 3, mercury ion antigen
One, utilizes mercury-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antigen prepd antibody
The Bal b/C small white mouse of (1) getting age in 8-10 week is as laboratory animal.Fundamental immunity dosage is the 0.25-4.0mg/kg body weight in the experiment, and booster immunization dosage is the 0.5-4.0mg/kg body weight.
(2) fundamental immunity: use PBS with the mercury-p-aminophenyl methyl EDTA-BSA antigen diluent of the foregoing description 1 preparation solution as 1mg/mL; Getting the 1mL antigenic solution filters with sterilizing filter; Add the Freund's complete adjuvant of 1mL then, fully emulsified, indiffusion in splashing into water.The antigen that emulsification is good adopts abdominal cavity and back subcutaneous multi-point injection Bal b/C mouse, and the ID of every mouse is 0.1mg (the about 23-25g of Bal b/C mouse body weight in 8 ages in week).Fundamental immunity is only carried out once.
(3) booster immunization: fundamental immunity 3-4 gets the good mercury of the above-mentioned dilution of 1mL-p-aminophenyl methyl EDTA-BSA antigenic solution after week, filters with sterilizing filter, adds the 1mL Freund's incomplete adjuvant then, and is fully emulsified, indiffusion in splashing into water.The antigen that emulsification is good adopts abdominal cavity and back subcutaneous multi-point injection Bal b/C mouse, and the ID of every mouse is 0.1mg (the about 25-27g of Bal b/C mouse body weight this moment).
Whenever according to the method described above, at a distance from 3-4 week booster immunization once beginning from booster immunization for the third time, takes a blood sample from mouse orbit in each immunity back 7-10 days; Measure antibody titer, wait to tire, put to death mouse greater than after 1: 4000; Antiserum(antisera) is isolated in blood sampling, promptly obtains antibody.
Two, antibody effect detection
Various damping fluids used in the following experiment are following:
(1) encapsulates damping fluid: the carbonate buffer solution of 0.05M, pH9.6;
(2) PBS: the 0.1M, the pH7.5 phosphate buffered saline buffer that contain the NaCl of 0.9% quality percentage composition;
(3) sample diluting liquid PBSTG: contain the tween 20 of 0.1% volumn concentration and the PBS solution that final concentration is the 1g/L gelatin;
(4) Citrate trianion-phosphate buffered saline buffer: contain 0.01M trisodium citrate and 0.03M Na
2HPO
4The aqueous solution of pH5.5;
(5) substrate buffer solution: contain in Citrate trianion-phosphate buffered saline buffer that final concentration is 2.0mg/mL O-Phenylene Diamine (OPD) at 10.0mL that to add 4 μ L volumn concentrations be 30% H
2O
2The solution that obtains;
(6) sulphuric acid soln of stop buffer: 2.0M;
(7) washings: contain 8.0g NaCl, 0.2g KH in every 1L washings
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20, all the other are water.
The antibody (with the antibody of mercury-p-aminophenyl methyl EDTA-BSA immune mouse preparation) of above-mentioned steps one preparation is detected as follows; With the antigen of preparation among antigen and the embodiment 1 of preparation among the embodiment 2 and the antibody of preparation thereof is example, below the concrete experimentation of detailed description:
(1) antibody suppresses experiment
1, the preparation of Hg-p-aminophenyl methyl EDTA-OVA envelope antigen solution
After the Hg-p-aminophenyl methyl EDTA-OVA antigen of the foregoing description 2 preparation thawed fully, with the above-mentioned damping fluid that encapsulates by carrying out gradient dilution in 1: 500,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000.
2, the preparation of EDTA-Hg inner complex standard solution
(1) takes by weighing 29.22mg EDTA, fully be dissolved in the 10mL zero(ppm) water, regulate pH to 10.0 with 1M NaOH;
(2) take by weighing 27.15mg HgCl
2, be dissolved in the zero(ppm) water, and in the EDTA solution of (1) preparation that dropwise joins above-mentioned steps, magnetic agitation, fully huge legendary turtle is closed;
(3) solution with step (2) is settled to 20.0mL, and obtaining final concentration is the EDTA-Hg solution of 2.46mg/mL, and wherein Hg ionic concentration is 1mg/mL;
(4) with sample diluting liquid the EDTA-Hg solution of above-mentioned steps (3) is made into the EDTA-Hg inner complex standard solution that concentration is 2000ng/mL (with the actual cubage of Hg solution ion).
3, the preparation of Hg-p-aminophenyl methyl EDTA-BSA antiserum(antisera) diluent
With the Hg-p-aminophenyl methyl EDTA-BSA antiserum(antisera) of above-mentioned steps one preparation with sample diluting liquid by carrying out gradient dilution in 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000.
4, use the mixed solution of forming by PBS and glycerine (volume ratio of PBS and glycerine is 1: 1) to be diluted to 0.1mg/mL IgG-HRP, be stored in-20 ℃ of refrigerators.Dilute by 1: 1000 with sample diluting liquid during use.
5, the checker of antigen, antibody experiment
(1) encapsulating of coating antigen: the envelope antigen solution of the different concns of above-mentioned steps 1 preparation is joined in the enzyme plate every hole 100 μ L, 37 ℃ of incubations 3 hours; Remove the solution in the enzyme plate, wash plate 4 times, dry with washings;
(2) in the enzyme plate of step (1), add the EDTA-Hg inner complex standard solution (experimental port) of above-mentioned steps 2 preparations, every hole 50 μ L do not add EDTA-Hg inner complex standard solution in the control wells and add 50 μ L sample diluting liquids;
The antiserum(antisera) diluent of the different concns that (3) adding above-mentioned steps 3 prepares in above-mentioned experimental port and control wells respectively, every hole 50 μ L; 37 ℃ of incubations 30 minutes; Outwell the solution in the enzyme plate, wash plate 4 times, dry with washings;
(4) in experimental port and control wells, add the IgG-HRP of 100 μ L respectively, 37 ℃ of incubations 30 minutes with sample diluting liquid dilution; Wash plate 4 times with washings, outwell the solution in the enzyme plate, dry;
(5) in experimental port and control wells, add 100 μ L substrate buffer solutions respectively, 37 ℃ of incubations add the sulphuric acid soln termination reaction of 50 μ L 2.0M after 15 minutes again in every hole;
(6) under 492nm, measure light absorption value.
3 repetitions are established in experiment, get the MV of three experimental results, and the result is as shown in table 1.
Table 1, antigen, antibody checker experimental result
In the table 1,
aEDTA-Hg competition colour developing value A is not added in expression
0,
bThe EDTA-Hg competition colour developing value A of 50 μ L 2000ng/mL is added in expression
2000
The result shows, when envelope antigen and antiserum(antisera) concentration are suitable, the inhibition phenomenon just arranged, and promptly the absorbance in 2000ng/mL hole and 0ng/mL hole has difference, and 2000ng/mL hole absorbance is little, 0ng/mL hole absorbance height; Calculate the best of breed of antigen, antibody with inhibiting rate, from table 1, can find out, when the envelope antigen extent of dilution is 1: 8000, the antiserum(antisera) extent of dilution is 1: 2000 o'clock, and the inhibiting rate of this moment is best, is 92.1% (inhibiting rate=(A
0-A
2000)/A
0), also promptly the inhibition effect of this moment is best.The Hg-p-aminophenyl methyl EDTA-BSA that the foregoing description 1 preparation is described can be used as the antibody that immunogen preparing goes out to detect mercury ion.
(2) foundation of EDTA-Hg typical curve
The EDTA-Hg inner complex standard solution of above-mentioned steps () preparation is diluted to following different concentration: 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL respectively with sample diluting liquid.
(1) encapsulating of coating antigen: the Hg-p-aminophenyl methyl EDTA-OVA antigen of the foregoing description 2 preparation is joined in the enzyme plate every hole 100 μ L, 37 ℃ of incubations 3 hours after according to dilution in 1: 8000; Remove the solution in the enzyme plate, wash plate 4 times, dry with washings;
(2) in the enzyme plate of step (1), add the EDTA-Hg inner complex standard solution (experimental port) of above-mentioned different concns respectively, every hole 50 μ L do not add EDTA-Hg inner complex standard solution in the control wells and add 50 μ L sample diluting liquids;
(3) extension rate that in above-mentioned experimental port and control wells, adds in the above-mentioned steps () 3 preparations respectively is 1: 2000 an antiserum(antisera) diluent, every hole 50 μ L; 37 ℃ of incubations 30 minutes; Outwell the solution in the enzyme plate, wash plate 4 times, dry with washings;
(4) in experimental port and control wells, adding 100 μ L extension rates respectively is 1: 1000 IgG-HRP, 37 ℃ of incubations 30 minutes; Wash plate 4 times with washings, outwell the solution in the enzyme plate, dry;
(5) in experimental port and control wells, add 100 μ L substrate buffer solutions respectively, 37 ℃ of incubations add the sulphuric acid soln termination reaction of 50 μ L 2.0M after 15 minutes again in every hole;
(6) under 492nm, measure light absorption value;
(7) drawing standard curve: with the EDTA-Hg inner complex standard solution of different concns (ng/mL) as the X axle, with the ratio (B/B of absorbance
0* 100%, wherein, B is the mean light absorbency value of EDTA-Hg inner complex standard solution, B
0Mean light absorbency value for control wells) as the Y axle, the drawing standard graphic representation.
3 repetitions are established in experiment, get the MV of three experimental results, and the canonical plotting that obtains is as shown in Figure 2.The result shows, its sensitivity (IC
50) be 134.1ng/mL, sensing range is 29.6ng/mL-2000ng/mL.Explain that the antibody that the Hg-p-aminophenyl methyl EDTA-BSA of the foregoing description 1 preparation obtains as the antigen immune mouse has good effect.
(3) antibodies specific detects
The preparation of EDTA-Hg inner complex standard solution
1, the preparation of the similar huge legendary turtle compound of EDTA-Hg
With reference to the preparation method of EDTA-Hg inner complex in the above-mentioned steps (), preparation EDTA-Cd, EDTA-Cu and EDTA-Pb supply the examination standard solution, calculate the concentration that each supplies the examination standard solution with each metals ion actual content in the solution.
With sample diluting liquid above-mentioned similar inner complex is diluted to following concentration: 20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL respectively.
2, set up typical curve separately, measure concentration IC in the inhibition
50(inhibiting rate reaches 50% standard specimen concentration value).
The establishment method of typical curve is identical with the establishment method of above-mentioned EDTA-Hg typical curve.
Cross reacting rate (%)=(EDTA-HgIC
50The similar huge legendary turtle compound of)/(EDTA-Hg IC
50) * 100%.
3 repetitions are established in experiment, get the MV of three experimental results, and the result is as shown in table 2.The result shows; The Hg-p-aminophenyl methyl EDTA-BSA antibody of above-mentioned steps one preparation and the cross reacting rate of the similar huge legendary turtle compound of other EDTA-Hg are very little, explain that the antibody of the Hg-p-aminophenyl methyl EDTA-BSA immune mouse generation for preparing with the foregoing description 1 has excellent specificity.
The specific detection of table 2Hg-p-aminophenyl methyl EDTA-BSA antibody
Analyte IC
50(ng/mL) cross reacting rate (%)
EDTA-Hg 134.1 100
EDTA-Cd ＞20000 ＜0.5
EDTA-Cu ＞20000 ＜0.5
EDTA-Pb ＞20000 ＜0.5
EDTA ＞20000 ＜0.5
Claims (10)
1. the preparation method of a mercury ion antigen may further comprise the steps:
(1) with the acid solution and the reaction of solubility nitrite of p-aminophenyl methylethylenediaminetetraacetic acid, obtains diazotizing p-aminophenyl methylethylenediaminetetraacetic acid;
(2) with the diazotizing p-aminophenyl methylethylenediaminetetraacetic acid and the carrier proteins reaction of above-mentioned steps (1), obtain coupled product;
(3) with the coupled product and the mercury ion reaction of above-mentioned steps (2), obtain mercury ion antigen;
The reaction conditions of said step (1) is: 0-5 ℃ of reaction under the lucifuge condition;
In the said step (1), the acid solution of said p-aminophenyl methylethylenediaminetetraacetic acid is that the p-aminophenyl methylethylenediaminetetraacetic acid is dissolved in hydrochloric acid, sulfuric acid, nitric acid, crosses the acid solution that obtains in chloric acid or the fluoroboric acid; In the said acid solution, hydrionic mol ratio is 1 in said p-aminophenyl methylethylenediaminetetraacetic acid and the said acid solution: (4-10);
In the said step (1), the mol ratio of said p-aminophenyl methylethylenediaminetetraacetic acid and said solubility nitrite is 1: (1-3);
In the said step (2), said carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or oralbumin; The mol ratio of said carrier proteins and said diazotizing p-aminophenyl methylethylenediaminetetraacetic acid is 1: (5-25);
The reaction conditions of said step (2) is: temperature of reaction is 15-25 ℃, and the pH value is 8.5-10.5, and the reaction times is 0.5-24h;
The reaction conditions of said step (3) is: temperature of reaction is 15-25 ℃, and the pH value is 8.5-10.5, and the reaction times is 0.5-24h;
In the said step (3), the mol ratio of said conjugate and said mercury ion is 1: (1-10).
2. method according to claim 1 is characterized in that: in the said acid solution, hydrionic mol ratio is 1: 6 in said p-aminophenyl methylethylenediaminetetraacetic acid and the said acid solution;
The mol ratio of said p-aminophenyl methylethylenediaminetetraacetic acid and said solubility nitrite is 1: 2.
3. method according to claim 1 is characterized in that: the mol ratio of said carrier proteins and said diazotizing p-aminophenyl methylethylenediaminetetraacetic acid is 1: 10.
4. method according to claim 1 is characterized in that: in the said step (3), the mol ratio of said conjugate and said mercury ion is 1: 5.
5. method according to claim 1 is characterized in that: the reaction conditions of said step (1) is: the following 4 ℃ of reactions of lucifuge condition.
6. method according to claim 1 is characterized in that: the reaction conditions of said step (2) is: temperature of reaction is 25 ℃, and the pH value is 9.5, and the reaction times is 12h;
The reaction conditions of said step (3) is: temperature of reaction is 25 ℃, and the pH value is 9.5, and the reaction times is 12h.
7. method according to claim 1 is characterized in that: in the said step (2), before said carrier proteins and the said diazotizing p-aminophenyl methylethylenediaminetetraacetic acid reaction, earlier said carrier proteins is dissolved in the carbonate buffer solution; The pH of said carbonate buffer solution is 9.0-9.6.
8. method according to claim 7 is characterized in that: in the said step (2), the pH of said carbonate buffer solution is 9.5.
9. according to claim 7 or 8 described methods, it is characterized in that: in the said step (2), also comprise the step that said conjugate is dialysed;
In the said step (3), also comprise the step that said mercury ion antigen is dialysed.
10. adopt the mercury ion antigen that arbitrary described method obtains among the claim 1-9.
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| CN200910083947A CN101885768B (en) | 2009-05-11 | 2009-05-11 | Mercury ion antigens and preparation method and application thereof |
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Non-Patent Citations (4)
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| James B. Delehanty 等.Identification of Important Residues in Metal-Chelate Recognition by Monoclonal Antibodies.《Biochemistry》.2003,第42卷(第48期),14173-14183. * |
| 杨凤丽 等.重金属汞单克隆抗体的制备及鉴定.《高技术通讯 》.2005,第18卷(第5期),531-536. * |
| 黄佳俊 等.重金属汞螯合物人工抗原的合成与鉴定.《中国生物工程杂志》.2008,第28卷(第2期),71-75. * |
| 黄峙.重金属-螯合剂复合物McAb制备及免疫检测技术进展.《卫生研究》.2004,第33卷(第6期),762-764. * |
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