CN102190723A - Chromium ion antigen and preparation method and application thereof - Google Patents
Chromium ion antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a chromium ion antigen and a preparation method and application thereof. The method for preparing the chromium ion antigen comprises the following steps of: (1) performing diazotization reaction on a chelant to obtain a diazotized chelant; (2) performing coupling reaction on the diazotized chelant obtained in the step (1) and a carrier protein to obtain a coupled product; and (3) performing complex reaction on the coupled product obtained in the step (2) and chromium ions to obtain a complex, namely the chromium ion antigen. By the method for preparing the chromium ion antigen, the chromium antigen can be convenient and quick to obtain; moreover, the synthetic steps are concise, the synthetic cost is low and the effect is good. An antigen obtained by immunizing through the chromium ion antigen prepared by the method has high specificity and low minimum detection limit. Therefore, the method for preparing the chromium ion antigen and the chromium antigen prepared by the method have wide application prospect in enzyme-linked immunosorbent assay of chromium ions.
Description
Technical field
The present invention relates to chromium ion antigen and preparation method thereof and application.
Background technology
Chromium is one of pollution substance important in the samples such as domestic and international food, medicine, environment.Heavy metal chromium mainly exists with trivalent chromium and chromic form in environment.Wherein, chromic toxicity is more much higher than trivalent chromium, to mucocutaneous have stimulate and corrosive nature, and easier for absorption of human body, accumulate, be confirmed to be carcinogens, therefore, the intake and the valence state thereof of heavy metal chromium have a significant impact HUMAN HEALTH.The compound of heavy metal chromium is widely used in industries such as process hides, metallurgy, textile production and chromium plating, chromium content in the environment is in continuous increase, therefore, heavy metal chromium also becomes must survey element in various countries' environmental monitoring, and chromic measuring method also is subjected to environmental monitoring person's extensive concern.
The measuring method of chromium is more, mainly contains spectrophotometry, chemoluminescence method, atomic absorption spectrometry, fluorometry, electrochemical methods and ion chromatography etc.The content that these several instrument detecting methods detect heavy metal chromiums not only needs expensive plant and instrument, and the testing cost height, detection time is long and it is numerous and diverse to detect step, can not be used for on-the-spot rapid detection.Immunoassay technology has been widely used in the environmental pollution of toxin, sterilant, explosive etc. and the detection of hazardous material, compare with other detection systems, immunologic detection method has fast, cheap, easy, sensitive and special advantage, can portablely carry out the scene and detect.Immunity detection reagent can a certain predetermined substance of rapid detection content, detect the heavy metal chromium ion but immunoassay will be applied to, must preparation corresponding antigen and antibody.
Summary of the invention
An object of the present invention is to provide the antigenic method of a kind of preparation chromium ion.
The antigenic method of preparation chromium ion provided by the present invention comprises the steps:
(1) sequestrant is carried out diazotization reaction, obtain the diazotization sequestrant;
(2) diazotizing sequestrant and the carrier proteins that described step (1) is obtained carries out linked reaction, obtains coupled product;
(3) coupled product and the chromium ion that described step (2) is obtained carries out complex reaction, obtains complex compound, promptly obtains chromium ion antigen.
The method of diazotization reaction may further comprise the steps described in the described step (1):
Described sequestrant is dissolved in the aqueous acid, obtains solution I; In described solution I, add the nitrite aqueous solution again, obtain solution II; Is 0 ℃-5 ℃ or 0 ℃ or 4 ℃ or 5 ℃, lucifuge and reaction times to be to react under the condition of 15min with described solution II in temperature, promptly obtains described diazotization sequestrant.
The molar ratio of carrier proteins and described diazotization sequestrant is 1 described in the described step (2): (5-25) or 1: 10 or 1: 5 or 1: 25;
Described linked reaction is to be 4 ℃-10 ℃ or 4 ℃ or 7 ℃ or 10 ℃ and reaction times to be to carry out under the condition of 4h-24h or 12h or 4h or 24h in temperature;
Described carrier proteins is bovine serum albumin or oralbumin or human serum albumin or hemocyanin.
The molar ratio of coupled product and described chromium ion is 1 described in the described step (3): (1-10) or 1: 3 or 1: 1 or 1: 10;
Described complex reaction be temperature be 4 ℃-25 ℃ or 25 ℃ or 4 ℃ or 15 ℃, pH value for 7.0-8.0 7.0 or 7.5 or 8.0 and the reaction times be to carry out under the condition of 4h-24h or 12h or 4h or 24h.
The molar ratio of sequestrant described in the described step (1) and described acid is 1: (4-10) or 1: 10 or 1: 7 or 1: 4;
The molar ratio of described sequestrant and described nitrite is 1: (1-3) or 1: 2 or 1: 1 or 1: 3;
Described sequestrant is the p-aminophenyl methylethylenediaminetetraacetic acid;
Described acid is hydrochloric acid or sulfuric acid or nitric acid or crosses chloric acid or fluoroboric acid;
Described nitrite is a Sodium Nitrite.
Linked reaction described in the described step (2) also comprises dialyses described coupled product, obtains the step of the coupled product of purifying;
Described carry out linked reaction before, also comprise described carrier proteins is dissolved in step in the carbonate buffer solution.
In the described step (3), also comprise the step that described complex compound is dialysed.
The chromium ion antigen for preparing according to described method also belongs to protection scope of the present invention.
The antibody that is obtained by described chromium ion antigen prepd also belongs to protection scope of the present invention.
The application in detecting chromium ion of described chromium ion antigen and/or described antibody also belongs to protection scope of the present invention.
The antigenic method of preparation chromium ion provided by the present invention can obtain chromium ion antigen quickly and easily, and synthesis step is short and sweet, synthetic cost is low, effective.Carry out with the chromium 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.Therefore, antigenic method of preparation chromium ion provided by the present invention and the chromium ion antigen that is obtained by this method will have broad application prospects in the enzyme linked immunosorbent detection of chromium ion.
Description of drawings
Fig. 1 is the antigenic synthetic route chart of chromium ion.
Fig. 2 is for EDTA-Cr being the chromium ion indirect elisa method typical curve that standard model is set up.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
P-aminophenyl methylethylenediaminetetraacetic acid (available from Sigma company, cat. no is A3473); Sheep anti-mouse igg-HRP (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 (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), CrCl
36H
20 (available from Sigma company, cat. no is 27096); All the other HCl, K
2CO
3, NaNO
2With conventional reagent such as glycerine all available from Beijing chemical reagents corporation.
Embodiment 1, chromium ion antigen and preparation thereof
One, the antigenic preparation of chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin (Cr-p-aminophenyl methyl EDTA-BSA)
Method I
The antigenic synthetic route chart of chromium ion as shown in Figure 1.
1, the diazotization of p-aminophenyl methylethylenediaminetetraacetic acid
(1) takes by weighing solid p-aminophenyl methylethylenediaminetetraacetic acid 1.985mg, fully be dissolved in the HCl aqueous solution of 0.5mL 0.1M, obtain solution I.In the solution I, the molar ratio of p-aminophenyl methylethylenediaminetetraacetic acid and HCl is 1: 10.
(2) solution I that above-mentioned steps (1) is obtained stirs under 4 ℃ of lucifuge conditions, and every interval 30s drips 30 μ L quality percentage compositions in solution I be 1% NaNO
2The aqueous solution obtains solution II.NaNO
2Add-on be excessive, i.e. p-aminophenyl methylethylenediaminetetraacetic acid and NaNO
2Molar ratio be 1: 2.With the starch-kalium iodide test paper solution II that above-mentioned steps (2) obtains is tested, after test paper becomes basket, reacted 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 bovine serum albumin (BSA) 33.0mg, fully be dissolved in the carbonate buffer solution of 2.0mL 0.05M, obtain solution III.
0.05M the carbonate buffer solution compound method as follows:
With 1.5gNa
2CO
3And 2.93gNaHCO
3Be dissolved in the distilled water, be settled to 1000mL with distilled water, the pH value is 9.6.
(2) the diazotization p-aminophenyl methylethylenediaminetetraacetic acid solution that above-mentioned steps 1 is obtained dropwise adds in the solution III of above-mentioned steps (1), and making the molar ratio of BSA and diazotization p-aminophenyl methylethylenediaminetetraacetic acid is 1: 10, obtains solution IV.
(3) linked reaction: under 4 ℃, the solution IV stirring of above-mentioned steps (2) is spent the night (12 hours), obtain coupled product solution.
(4) linked reaction is after 12 hours, the coupled product solution that above-mentioned steps (3) is obtained places dialysis tubing, with pH is 7.5 phosphate buffered saline buffer dialysis 2 days, changes dialyzate every day 3 times, obtains the diazotization p-aminophenyl methylethylenediaminetetraacetic acid of purifying and the conjugate of carrier proteins.
Above-mentioned pH is that the compound method of 7.5 phosphate buffered saline buffers is as follows:
With 8.0g NaCl, 0.2g KH
2PO
4With 2.96g Na
2HPO
412H
2O is dissolved in the distilled water, is settled to 1000mL with distilled water again.
3, the complexing of chromium ion
(1) takes by weighing 4.00mg CrCl
36H
2O is mixed with 0.1M CrCl with ultrapure water
3The aqueous solution.The diazotization p-aminophenyl methylethylenediaminetetraacetic acid of the purifying that step 2 is obtained and the conjugate solution of carrier proteins are adjusted to 7.5 with the HCl of 1M with its pH value, obtain adjusting the conjugate solution after the pH value.
(2) with the CrCl of above-mentioned steps (1)
3The aqueous solution dropwise joins in the conjugate solution after the adjustment pH value that above-mentioned steps (1) obtains, the stirring while dripping, and making the molar ratio of diazotization p-aminophenyl methylethylenediaminetetraacetic acid and chromium ion is 1: 3, obtains solution V.
(3) be to stir 12 hours under 7.5 the condition the solution V of above-mentioned steps (2) at 25 ℃, pH value, with pH is 7.5 phosphate buffered saline buffer dialysis 2 days, change dialyzate every day 3 times, to remove chromium ion and other impurity that does not connect, the dialysis product is chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin (Cr-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 standby to place-20 ℃ of refrigerators to preserve.
Method II
1, the diazotization of p-aminophenyl methylethylenediaminetetraacetic acid
(1) takes by weighing solid p-aminophenyl methylethylenediaminetetraacetic acid 1.985mg, fully be dissolved in the HCl aqueous solution of 0.35mL 0.1M, obtain solution I.In the solution I, the molar ratio of p-aminophenyl methylethylenediaminetetraacetic acid and HCl is 1: 7.
(2) divided by outside the following method, all the other methods are all identical with method I:
A, the solution I that above-mentioned steps (1) is obtained stir under 0 ℃ of lucifuge condition;
B, every interval 30s drip 15 μ L quality percentage compositions in solution I be 1% NaNO
2The aqueous solution obtains solution II.NaNO
2Add-on be excessive, i.e. p-aminophenyl methylethylenediaminetetraacetic acid and NaNO
2Molar ratio be 1: 1.
2, diazotization p-aminophenyl methylethylenediaminetetraacetic acid and carrier proteins carry out coupling
(1) identical with method I.
(2) molar ratio except that BSA and diazotization p-aminophenyl methylethylenediaminetetraacetic acid is 1: 5, and all the other methods are all identical with method I.
(3) linked reaction: under 7 ℃, the solution IV stirring 4h with above-mentioned steps (2) obtains coupled product solution.
(4) identical with method I.
3, the complexing of chromium ion
(1) the conjugate solution of the diazotization p-aminophenyl methylethylenediaminetetraacetic acid of the purifying that step 2 is obtained and carrier proteins is adjusted to its pH value outside 7.0 with the HCl of 1M, and all the other methods are all identical with method I.
(2) molar ratio except that diazotization p-aminophenyl methylethylenediaminetetraacetic acid and chromium ion is 1: 1, and all the other methods are all identical with method I.
(3) removing the solution V of above-mentioned steps (2) is to stir 4h under 7.0 the condition at 4 ℃, pH value, and all the other methods are all identical with method I.
(4) identical with method I.
Method III
1, the diazotization of p-aminophenyl methylethylenediaminetetraacetic acid
(1) takes by weighing solid p-aminophenyl methylethylenediaminetetraacetic acid 1.985mg, fully be dissolved in the HCl aqueous solution of 0.2mL 0.1M, obtain solution I.In the solution I, the molar ratio of p-aminophenyl methylethylenediaminetetraacetic acid and HCl is 1: 4.
(2) divided by outside the following method, all the other methods are all identical with method I:
A, the solution I that above-mentioned steps (1) is obtained stir under 5 ℃ of lucifuge conditions;
B, every interval 30s drip 45 μ L quality percentage compositions in solution I be 1% NaNO
2The aqueous solution obtains solution II.NaNO
2Add-on be excessive, i.e. p-aminophenyl methylethylenediaminetetraacetic acid and NaNO
2Molar ratio be 1: 3.
2, diazotization p-aminophenyl methylethylenediaminetetraacetic acid and carrier proteins carry out coupling
(1) identical with method I.
(2) molar ratio except that BSA and diazotization p-aminophenyl methylethylenediaminetetraacetic acid is 1: 25, and all the other methods are all identical with method I.
(3) linked reaction: under 10 ℃, the solution IV stirring 24h with above-mentioned steps (2) obtains coupled product solution.
(4) identical with method I.
3, the complexing of chromium ion
(1) the conjugate solution of the diazotization p-aminophenyl methylethylenediaminetetraacetic acid of the purifying that step 2 is obtained and carrier proteins is adjusted to its pH value outside 8.0 with the HCl of 1M, and all the other methods are all identical with method I.
(2) molar ratio except that diazotization p-aminophenyl methylethylenediaminetetraacetic acid and chromium ion is 1: 10, and all the other methods are all identical with method I.
(3) except that the solution V of above-mentioned steps (2) is stirred the 24h under 15 ℃, pH value are 8.0 condition, all the other methods are all identical with method I.
(4) identical with method I.
Two, the antigenic preparation of chromium-p-aminophenyl methylethylenediaminetetraacetic acid-oralbumin (Cr-p-aminophenyl methyl EDTA-OVA)
Method is identical with immunogenic preparation method described in the experiment one, and different is that used carrier proteins is oralbumin (OVA).
Embodiment 2, the antigenic application of chromium ion
One, utilizes chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antigen prepd antibody
(1) gets the Bal b/C small white mouse (available from Military Medical Science Institute's Experimental Animal Center) in age in 6-8 week as laboratory animal.
(2) fundamental immunity: is the solution of 1mg/ml with PBS with chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antigen diluent that the foregoing description 1 prepares, getting the 1ml antigenic solution filters with sterilizing filter, the Freund's complete adjuvant that adds 1ml then, fully emulsified, indiffusion in splashing into water.Antigen that emulsification is good adopts abdominal cavity and back subcutaneous multi-point injection Bal b/C mouse, and injected dose is a 0.1mg antigen/only.
(3) booster immunization: fundamental immunity is after 3 weeks, get the good chromium of the above-mentioned dilution of 1ml-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antigenic solution, filter, add the 1ml Freund's incomplete adjuvant then with sterilizing filter, fully emulsified, indiffusion in splashing into water.Antigen that emulsification is good adopts abdominal cavity and back subcutaneous multi-point injection Balb/C mouse, and injected dose is a 0.1mg antigen/only.
According to the method described above once every 2 all booster immunizations, totally 4 times, and from booster immunization for the third time, antibody titer from the mouse orbit blood sampling, is measured in each immunity back the 5th day, wait to tire greater than after 1: 8000, chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antiserum(antisera) is isolated in blood sampling, promptly obtains antibody.
Two, antibody effect detection
Various damping fluids used in the following experiment are as follows:
(1) bag is cushioned the carbonate buffer solution of liquid: 0.05M, pH9.6;
0.05M, the compound method of the carbonate buffer solution of pH9.6 is as follows:
Take by weighing 1.5g Na respectively
2CO
3With 2.93g NaHCO
3, after fully dissolving with distilled water, be settled to 1000mL with distilled water.
(2) phosphate buffered saline buffer: the 0.1M, the pH7.5 phosphate buffered saline buffer that contain the quality percentage composition and be 0.9% NaCl.
The compound method of described phosphate buffered saline buffer is as follows:
Take by weighing 8.0g NaCl, 0.2g KH respectively
2PO
4With 2.96g Na
2HPO
412H
2O after fully dissolving with distilled water, is settled to 1000mL with distilled water.
(3) sample diluting liquid PBSTG: contain 8.0g NaCl, 0.2g KH in every 1L sample diluting liquid
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20 and 1g gelatin, all the other are water.
(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: 20.0mg O-Phenylene Diamine (OPD) is dissolved in 10.0mL Citrate trianion-phosphate buffered saline buffer, adds 4 μ L volumn concentrations then and be 30% H
2O
2The solution that obtains;
(6) aqueous sulfuric acid 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 and 1.0mL Tween-20, all the other are water.
(1) antibody suppresses experiment
1, the preparation of chromium-p-aminophenyl methylethylenediaminetetraacetic acid-oralbumin envelope antigen solution
With after chromium-p-aminophenyl methylethylenediaminetetraacetic acid-oralbumin antigen thaws fully of the foregoing description 1 preparation, be cushioned liquid by carrying out gradient dilution in 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000 with above-mentioned bag.
2, the preparation of EDTA-Cr inner complex standard solution
(1) takes by weighing 14.32mg EDTA-Na
2, fully be dissolved in the 5mL distilled water, obtaining concentration is the EDTA aqueous solution of 2.864mg/mL.
(2) take by weighing 10.24mg CrCl
36H
2O is dissolved in the distilled water, and in the EDTA aqueous solution of (1) preparation that dropwise joins above-mentioned steps, magnetic agitation, fully huge legendary turtle is closed, and obtains the solution of EDTA and Cr ion chelating product.
(3) EDTA of step (2) and the solution of Cr ion chelating product are settled to 10.0mL, obtaining the Cr ionic concn is the solution of the EDTA-Cr chelating product of 1mg/mL.
(4) with sample diluting liquid the EDTA-Cr solution of above-mentioned steps (3) is made into the EDTA-Cr inner complex standard solution that concentration is 2000ng/mL (with the actual cubage of Cr ion in the solution).
3, the preparation of chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin antiserum(antisera) diluent
With the chromium-p-aminophenyl methylethylenediaminetetraacetic acid-bovine serum albumin 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, sheep anti-mouse igg-HRP is used the mixed solution of forming by PBS and glycerine (volume ratio of PBS and glycerine is 1: 1) be diluted to 0.1mg/mL, be stored in-20 ℃ of refrigerators.Dilute by 1: 1000 with sample diluting liquid during use.
5, the checker of antigen, antibody experiment
(1) the bag quilt 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) add the EDTA-Cr inner complex standard solution (experimental port) of above-mentioned steps 2 preparations in the enzyme plate of step (1), every hole 50 μ L do not add EDTA-Cr inner complex standard solution in the control wells and add 50 μ L sample diluting liquids;
(3) the antiserum(antisera) diluent of the different concns that 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) add 100 μ L substrate buffer solutions respectively in experimental port and control wells, 37 ℃ of incubations add 50 μ L stop buffer termination reactions after 15 minutes again in every hole;
(6) under 492nm, measure light absorption value.
3 repetitions are established in experiment, get the mean value of three experimental results, and the result is as shown in table 1.
Table 1 antigen and antibody checker experimental result
In the table 1,
aEDTA-Cr competition colour developing value A is not added in expression
0,
bThe EDTA-Cr 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, as can be seen from Table 1, when the envelope antigen extent of dilution is 1: 4000, the antiserum(antisera) extent of dilution is 1: 4000 o'clock, and the inhibiting rate of this moment is best, is 87.38% (inhibiting rate=(A
0-A
2000)/A
0* 100%), also promptly the inhibition effect of this moment is best.The Cr-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 chromium ion.
(2) foundation of EDTA-Cr typical curve
The EDTA-Cr inner complex standard solution of above-mentioned steps () preparation is diluted to following different concentration respectively with sample diluting liquid: 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL.
(1) the bag quilt of coating antigen: the chromium-p-aminophenyl methylethylenediaminetetraacetic acid-oralbumin 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: 4000; Remove the solution in the enzyme plate, wash plate 4 times, dry with washings;
(2) add the EDTA-Cr inner complex standard solution (experimental port) of above-mentioned different concns respectively in the enzyme plate of step (1), every hole 50 μ L do not add EDTA-Cr inner complex standard solution in the control wells and add 50 μ L sample diluting liquids;
(3) extension rate that adds preparation in the above-mentioned steps () respectively in above-mentioned experimental port and control wells is 1: 4000 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) adding 100 μ L extension rates respectively in experimental port and control wells 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) add 100 μ L substrate buffer solutions respectively in experimental port and control wells, 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-Cr inner complex standard solution of different concns (ng/mL) as X-axis, with the ratio (B/B of absorbance
0* 100%, wherein, B is the mean light absorbency value of EDTA-Cr inner complex standard solution, B
0Mean light absorbency value for control wells) as Y-axis, the drawing standard graphic representation.
3 repetitions are established in experiment, get the mean value of three experimental results, and the canonical plotting that obtains as shown in Figure 2.The result shows, its sensitivity (IC
50) be 93.88ng/mL, sensing range is 15.6ng/mL-2000ng/mL.Show the antibody that makes by antigen of the present invention effective.
(3) antibodies specific detects
1, the preparation of the similar huge legendary turtle compound of EDTA-Cr
With reference to the preparation method of EDTA-Cr inner complex in the above-mentioned steps (), the EDTA inner complex of preparation Cd, Cu, Hg, Zn, Mn, Fe, Mg metal calculates each concentration for the examination standard solution for the examination standard solution with each metal 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-Cr typical curve.
Cross reacting rate (%)=(EDTA-Cr IC
50The similar huge legendary turtle compound of)/(EDTA-Cr IC
50) * 100%.
3 repetitions are established in experiment, and the result takes the mean, and the result is as shown in table 2.Table 2 shows that the cross reacting rate of the Cr-EDTA antibody that makes in the present embodiment step 1 and other metal ion EDTA inner complex is very little, and it is good to illustrate by the specificity of the antibody of antigen prepd of the present invention.
The specific detection of table 2Cd-EDTA antibody
| Analyte | IC 50(ng/mL) | Cross reacting rate (%) |
| Cr-EDTA | 93.876 | 100 |
| Cd-EDTA | ＞10000 | ＜1 |
| Zn-EDTA | ＞10000 | ＜1 |
| Hg-EDTA | ＞10000 | ＜1 |
| Mn-EDTA | ＞10000 | ＜1 |
| Cu-EDTA | ＞10000 | ＜1 |
| Fe-EDTA | ＞10000 | ＜1 |
| Mg-EDTA | ＞10000 | ＜1 |
| EDTA | 72548 | ＜1 |
Claims (10)
1. one kind prepares the antigenic method of chromium ion, comprises the steps:
(1) sequestrant is carried out diazotization reaction, obtain the diazotization sequestrant;
(2) diazotizing sequestrant and the carrier proteins that described step (1) is obtained carries out linked reaction, obtains coupled product;
(3) coupled product and the chromium ion that described step (2) is obtained carries out complex reaction, obtains complex compound, promptly obtains chromium ion antigen.
2. method according to claim 1 is characterized in that: the method for diazotization reaction may further comprise the steps described in the described step (1):
Described sequestrant is dissolved in the aqueous acid, obtains solution I; In described solution I, add the nitrite aqueous solution again, obtain solution II; Is 0 ℃-5 ℃ or 0 ℃ or 4 ℃ or 5 ℃, lucifuge and reaction times to be to react under the condition of 15min with described solution II in temperature, promptly obtains described diazotization sequestrant.
3. method according to claim 1 and 2 is characterized in that:
The molar ratio of carrier proteins and described diazotization sequestrant is 1 described in the described step (2): (5-25) or 1: 10 or 1: 5 or 1: 25;
Described linked reaction is to be 4 ℃-10 ℃ or 4 ℃ or 7 ℃ or 10 ℃ and reaction times to be to carry out under the condition of 4h-24h or 12h or 4h or 24h in temperature;
Described carrier proteins is bovine serum albumin or oralbumin or human serum albumin or hemocyanin.
4. according to arbitrary described method among the claim 1-3, it is characterized in that:
The molar ratio of coupled product and described chromium ion is 1 described in the described step (3): (1-10) or 1: 3 or 1: 1 or 1: 10;
Described complex reaction be temperature be 4 ℃-25 ℃ or 25 ℃ or 4 ℃ or 15 ℃, pH value for 7.0-8.0 7.0 or 7.5 or 8.0 and the reaction times be to carry out under the condition of 4h-24h or 12h or 4h or 24h.
5. according to arbitrary described method among the claim 1-4, it is characterized in that:
The molar ratio of sequestrant described in the described step (1) and described acid is 1: (4-10) or 1: 10 or 1: 7 or 1: 4;
The molar ratio of described sequestrant and described nitrite is 1: (1-3) or 1: 2 or 1: 1 or 1: 3;
Described sequestrant is the p-aminophenyl methylethylenediaminetetraacetic acid;
Described acid is hydrochloric acid or sulfuric acid or nitric acid or crosses chloric acid or fluoroboric acid;
Described nitrite is a Sodium Nitrite.
6. according to arbitrary described method among the claim 1-5, it is characterized in that:
Linked reaction described in the described step (2) also comprises dialyses described coupled product, obtains the step of the coupled product of purifying;
Described carry out linked reaction before, also comprise described carrier proteins is dissolved in step in the carbonate buffer solution.
7. according to arbitrary described method among the claim 1-6, it is characterized in that:
In the described step (3), also comprise the step that described complex compound is dialysed.
8. the chromium ion antigen for preparing according to arbitrary described method among the claim 1-7.
9. the antibody that obtains by the described chromium ion antigen prepd of claim 8.
10. described chromium ion antigen of claim 8 and/or the described antibody of claim 9 application in detecting chromium ion.
Priority Applications (1)
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| CN2011100909788A CN102190723A (en) | 2011-04-12 | 2011-04-12 | Chromium ion antigen and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100909788A CN102190723A (en) | 2011-04-12 | 2011-04-12 | Chromium ion antigen and preparation method and application thereof |
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Cited By (4)
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| CN103439508A (en) * | 2013-08-25 | 2013-12-11 | 河南科技学院 | Indirect competitive enzyme linked immunosorbent assay kit for detecting chromium ions as well as preparation and detection methods thereof |
| CN105985439A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院上海生命科学研究院 | Nano antibody of specific identification heavy metal Cr and application of nano antibody |
| US9952471B2 (en) | 2015-06-25 | 2018-04-24 | Shenzhen China Star Optoelecrtronics Technology Co., Ltd. | Pixel electrode and liquid crystal display panel |
| CN108680737A (en) * | 2018-05-25 | 2018-10-19 | 清华大学深圳研究生院 | The detection kit of heavy metal chromium ion and its application |
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| CN101445557A (en) * | 2008-12-16 | 2009-06-03 | 中国农业大学 | Cadmium ion antigen and preparation method and application thereof |
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| SASAKI K等: "Monoclonal antibody to triva-lent chromium chelate complex and its application to measurement ofthe total chromium concentration", 《ANAL CHEM》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439508A (en) * | 2013-08-25 | 2013-12-11 | 河南科技学院 | Indirect competitive enzyme linked immunosorbent assay kit for detecting chromium ions as well as preparation and detection methods thereof |
| CN103439508B (en) * | 2013-08-25 | 2015-07-15 | 河南科技学院 | Indirect competitive enzyme linked immunosorbent assay kit for detecting chromium ions as well as preparation and detection methods thereof |
| CN105985439A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院上海生命科学研究院 | Nano antibody of specific identification heavy metal Cr and application of nano antibody |
| CN105985439B (en) * | 2015-02-04 | 2019-08-09 | 中国科学院上海生命科学研究院 | The nano antibody of specific recognition heavy metal Cr and its application |
| US9952471B2 (en) | 2015-06-25 | 2018-04-24 | Shenzhen China Star Optoelecrtronics Technology Co., Ltd. | Pixel electrode and liquid crystal display panel |
| CN108680737A (en) * | 2018-05-25 | 2018-10-19 | 清华大学深圳研究生院 | The detection kit of heavy metal chromium ion and its application |
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