CN101196521A - Indirectly racing ELISA detecting method for cadmium ion - Google Patents
Indirectly racing ELISA detecting method for cadmium ion Download PDFInfo
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- CN101196521A CN101196521A CNA2007100329146A CN200710032914A CN101196521A CN 101196521 A CN101196521 A CN 101196521A CN A2007100329146 A CNA2007100329146 A CN A2007100329146A CN 200710032914 A CN200710032914 A CN 200710032914A CN 101196521 A CN101196521 A CN 101196521A
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Abstract
The invention discloses an ELISA method by indirect competition for testing the cadmium ion, which is to add the extracting solution of the seafood samples and the standard fluid of cadmium ion in serial consistency into the millipore of the antigen pre-peridium strips, and then add the monoclonal antibody with the function of anti-cadmium ion into each millipore, blend for incubation, add the antibody with the function of anti-mouse IgG marked by HRP and then make incubation; stop the reaction after the substrate chromogenic cadmium ion, test the absorbency of each millipore by enzyme mark instrument, and calculate the contact in the samples in accordance with the curvilinear regression equation gained from the standard control. By utilizing the gained secretory positive cells of the monoclonal antibody with the function of anti-cadmium ion, the method provided in the invention can prepare in mass production the monoclonal antibody with the function of anti-cadmium ion. The method costs low, which can fast, simply and sensitively test the content of the cadmium ion in the samples. The invention can make fast and sensitive test on the heavy metal cadmium left in the seafood like fishes and shellfishes on a large scale.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of fast diagnosis method of the heavy-metal residual of aquatic product meat product, specifically is the indirect competitive ELISA method of utilizing the monoclonal antibody of preventing from heavy metal cadmium ion that cadmium ion is detected.
Background technology
Cadmium (Cdmium) atomic number 48 is one of heavy metals of serious harm human health, is mainly derived from cadmium ore deposit, cadmium smeltery.Of the same clan because of cadmium and zinc, normal and zinc symbiosis must have ZnO so smelt in the emission of zinc, CdO, and their high volatilities are that thousands of rice can be involved far in the center with pollution source.Along with developing rapidly of China's industrial or agricultural, the pollution of heavy metal is more and more serious in the environment.The murder by poisoning that heavy metal pollution causes exists lastingly, can circulate once more and enters food chain with soil, water body, food security is constituted a threat to the harm humans life and health.Cadmium poisoning can make renal function be damaged, and cadmium enters respiratory tract can cause that pneumonia, pulmonary emphysema act on digestive system and then cause enterogastritis.Cadmium poisoning person has excessive cadmium accumulation can make skeleton softening, distortion, fracture, atrophy usually with anaemia in the bone, also can cause cancer.The country of the heavy metal hygienic standard of limiting the quantity of all has qualification≤0.1mg/kg to cadmium content in the nuisanceless aquatic products safety requirements of agricultural product security quality (2001) in China's varieties of food items.European Union, the U.S., Japan, the leading exporter of China's aquatic products such as Korea S is also more and more stricter to the qualification of the content of beary metal of aquatic products, and rule and standard are also more and more harsher.
The detection method of heavy metal cadmium mainly contains at present:
The method of physics and chemistry: atomic absorption spectrophotometry (AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), inductivity coupled plasma mass spectrometry analysis (ICP-MS), atomic fluorescence spectrometry (AFS) etc.The characteristics of these methods are sensitive, accurately, but need complicated instrument and equipment, special analytical technology personnel, and standard items cost an arm and a leg, and the pre-treatment trouble can not detect a large amount of samples simultaneously, is not suitable for on-the-spot and applies on a large scale.
The immunology detection technology: present more application is enzyme-linked immuno-sorbent assay (ELISA) in the heavy metal cadmium detection, the principle of its detection is with cadmium ion and carrier protein couplet immune animal, acquisition is at the antibody of cadmium, utilizes in cadmium ion in the sample and the standard items cadmium ion competition binding antibody and with this cadmium in sample carried out qualitative or quantitative test.This method is easy, quick, can analyze batch samples simultaneously.
External at present immunology detection technology of having carried out heavy metal, successfully prepare In (III), Pb (II), Ni (II), Cd (II), Hg specificity heavy metal antibody such as (II), partial antibody has been applied to environment measuring, develops the elisa kit for detecting of heavy metal.The domestic report that the immunology detection of heavy metal is not arranged as yet, foreign literature report is insufficient, can not repeat synthetic artificial antigen according to it, and the enzyme joint inspection survey method of heavy metal of therefore developing the tool independent intellectual property right is significant.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, the object of the present invention is to provide a kind of indirect competitive ELISA method of utilizing heavy metal cadmium monoclonal antibody to detect cadmium ion, this method can be carried out quick, sensitive detection to heavy metal cadmium residual in the marine fish shellfish food on a large scale, ensures China's marine product edible safety and safeguards the interests of China in relevant domain of international trade.
Purpose of the present invention is achieved through the following technical solutions: a kind of detection method of cadmium ion indirect competitive ELISA comprises the steps: respectively the cadmium ion (Cd with fish and shellfish sample extracting solution and series concentration
2+) titer joins antigen and wrap by in the micropore of bar in advance, the monoclonal antibody that adds the Cadmium resistance ion again is to each micropore, and mixing is hatched, and adds the goat anti-mouse igg antibody of HRP mark in each micropore, hatches; Cessation reaction after substrate colour developing is measured the absorbance (OD) in each hole, the Regression Equations of reference standards gained, cadmium ion (Cd in the calculation sample with microplate reader
2+) content.
The detection method of above-mentioned cadmium ion indirect competitive ELISA comprises the steps:
(1) in antigen wraps by each micropore of bar in advance, all adds the 100mM EDTA (b diammonium edta) of 10 μ l respectively, in above-mentioned each micropore, add the Cd of 80 μ L fish and shellfish sample extracting solutions and 80 μ l series concentration then more respectively
2+Titer, mixing; Each micropore adds the monoclonal antibody of the Cadmium resistance ion of 1: 32000 times of dilution of 10 μ L then; Mixing is hatched 0.5～2h for 37 ℃; Wash micropore with cleansing solution then;
(2) each micropore adds the goat anti-mouse igg antibody of 100 μ L with the HRP mark of dilution dilution in 1: 4000, hatches 0.5～1h for 37 ℃; Wash micropore with cleansing solution then;
(3) each micropore add 100 μ L colour developing liquid TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid, reaction 10～20min;
(4) each micropore adds 50 μ L 2M H
2SO
4(stop buffer) cessation reaction, and vibration mixing;
(5) be determined at OD (absorbance) value under the 450nm wavelength with enzyme connection readout instrument;
(6) gained titer and sample absorbance being multiply by 100% again divided by the absorbance of 0 standard (titer of 0 concentration (ng/mL)), is ordinate with this titer calculated value, and the logarithm of concentration of cadmium ions (μ g/kg) is a horizontal ordinate drawing standard curve; B/B0 value according to each sample is read corresponding concentration of cadmium ions from typical curve, multiply by corresponding extension rate again, calculates concentration of cadmium ions actual in the sample.Described dilution and cleansing solution are the 0.01M phosphate buffer that PBS-T promptly contains 0.05% Tween-20.
In order to realize the present invention better, the pre-service as follows of fish and shellfish sample extracting solution: every gram flesh of fish or shellfish meat sample add the 2mL ethyl acetate, fully smash even matter; The centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; Mix extraction vortex 1min with equal-volume cyclohexane and distilled water, get the cyclohexane layer, every gram sample adds 12.5 μ L 1M HCl, and vortex 1min leaves standstill 30min again; Add the distilled water of 2 times of HCl volumes, 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH7.0～8.0 with 1N NaOH, after 10 times of diluted, promptly gets the fish and shellfish sample extracting solution.
The pre-bag of described antigen is prepared as follows by bar:
(1) bag quilt: to be cushioned liquid be that the 0.05M sodium carbonate buffer of pH9.6 is diluted to 0.2 μ g/mL with Cd-iEDTA-BSA with wrapping, and every micropore 100 μ L join in the polystyrene micropore plate hole, 4 ℃ of bags spent the night or 37 ℃ of bags by 2～3h; Described Cd-iEDTA-BSA is coupled to carrier protein BSA with iEDTA with cadmium ion and goes up acquisition;
(2) washing: after pouring out in the micropore plate hole bag and being cushioned liquid, 200 μ l cleansing solutions are added in each micropore; Pour out the liquid in the hole after 3～5 minutes once more, remove the liquid in the micropore fully; Described cleansing solution is for containing the 0.01M phosphate buffer (KH of 0.05% (mass percent) Tween-20
2PO
40.2g, Na
2PO
412H
2O 2.9g, NaCl 8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again);
(3) sealing: the every micropore of microwell plate add 150～200 μ L contain 0.5%～3% (W/V) BSA (bovine serum albumin(BSA)) 0.01M PBS (phosphate buffer, pH7.4), 37 ℃ of sealing 0.5h～1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% (W/V) sucrose phosphate buffer room temperature protection 3 hours;
(6) drying: after the microwell plate drying, promptly obtain antigen and wrap by bar in advance.
The monoclonal antibody of described Cadmium resistance ion is prepared from as follows: with iEDTA (is 1-(the different sulphur cyanobenzyl of 4-) vinyl diamines-N, N, N ', N '-tetraacethyl) cadmium ion is coupled on the hemocyanin (KLH), mix not formula Freund immunity BALB/c mouse, getting immune mouse spleen cell and SP2/0 myeloma cell merges, filter out the positive cell strain and the enlarged culture of stably excreting Cadmium resistance ion monoclonal antibody (MAb-Cd), the injection cell advances in the mouse body to induce ascites, and purifying obtains the monoclonal antibody of Cadmium resistance ion.
The indirect competitive ELISA method that above-mentioned cadmium ion detects can be applicable to detect marine fish shellfish food etc.
The present invention is by 96 hole ELISA Plate with the Cd-iEDTA-BSA bag, add cadmium ion titer (Cd-EDTA) and fish and shellfish sample extracting solution to be measured (X-EDTA), the monoclonal antibody that adds the Cadmium resistance ion again, envelope antigen Cd-iEDTA-BSA and free Cd-EDTA competition Cadmium resistance ion monoclonal antibody, Cd-EDTA that flush away is free and the compound of MAb-Cd-EDTA, the MAb that combines with envelope antigen Cd-iEDTA-BSA combines with the goat anti-mouse igg antibody of enzyme labeling again, cessation reaction after the substrate colour developing, measure the absorbance (OD) in each hole with microplate reader, the OD value is big more, Cd-EDTA content is few more freely in the sample, the Regression Equations of reference standards gained, Cd in the calculation sample
2+Content.
The present invention compared with prior art has following advantage and beneficial effect:
The positive cell of the secretion Cadmium resistance ion monoclonal antibody that utilization of the present invention obtains can make the monoclonal antibody of Cadmium resistance ion in a large number, and the method for being set up is with low cost, can be quick, easy, Cd in the working sample delicately
2+Content.Whole testing process approximately only needs 2h, does not need large-scale instrument and equipment and sensitivity can reach national examination criteria 100 μ g/kg samples.This method can be carried out quick, sensitive detection to heavy metal cadmium residual in the seashells food on a large scale, ensures China's marine product edible safety and safeguards the interests of China in relevant domain of international trade.
Description of drawings
Fig. 1 suppresses curve map for standard.
Fig. 2 is for suppressing the curve linear regression figure.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
The preparation of reagent:
Dilution and cleansing solution are the 0.01M phosphate buffer (KH that PBS-T promptly contains 0.05% Tween-20
2PO
40.2g, Na
2PO
412H
2O 2.9g, NaCl 8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again); Colour developing liquid be TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid; Stop buffer is that the 2M sulfuric acid solution is promptly measured the 111.2ml concentrated sulphuric acid (18M) dilution and is settled to 1000ml.
Embodiment 1
Utilize Cadmium resistance ion monoclonal antibody to detect the indirect competitive ELISA method of cadmium ion, comprise the steps:
(A) (be 1-(the different sulphur cyanobenzyl of 4-) vinyl diamines-N with iEDTA, N, N ', N '-tetraacethyl) cadmium ion is coupled on the hemocyanin (KLH), mix not formula Freund immunity BALB/c mouse, get immune mouse spleen cell and SP2/0 myeloma cell and merge, filter out the positive cell strain and the enlarged culture of stably excreting Cadmium resistance ion monoclonal antibody (MAb-Cd), the injection cell advances in the mouse body to induce ascites, and purifying obtains the monoclonal antibody of Cadmium resistance ion.
(B) with iEDTA cadmium ion is coupled to carrier protein BSA and goes up acquisition detection antigens c d-iEDTA-BSA.
(C) antigen is pre-wraps by the preparation of bar:
(1) bag quilt: (the 0.05M sodium carbonate buffer pH9.6) is diluted to Cd-iEDTA-BSA 0.2 μ g/mL, and every micropore 100 μ L join in the polystyrene micropore plate, and 4 ℃ of bags are spent the night to be cushioned liquid with bag.
(2) washing: after pouring out in the micropore plate hole bag and being cushioned liquid, microwell plate is upside down in pats on the thieving paper to guarantee to remove fully the liquid in the micropore.With wash bottle 200 μ l cleansing solutions are added in the hand-hole.Pour out the liquid in the micropore after 3 minutes once more, remove the liquid in the micropore fully.
(3) sealing: every micropore add 160 μ L contain 2% (W/V) BSA (Bovine serum album, bovine serum albumin(BSA)) 0.01M PBS (Phosphate-Buffered Saline, phosphate buffer, pH7.4), 37 ℃ the sealing 1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% (W/V) sucrose phosphate buffer room temperature protection 3 hours;
(6) drying: after putting the hothouse drying, obtain antigen and wrap by bar in advance, preserve in the packaging bag that contains drying agent of packing into.
(D) sample pretreatment (flesh of fish sample extracting solution)
Get the flesh of fish sample that shreds and place centrifuge tube, add the 2mL ethyl acetate, fully smash even matter by every gram sample; The centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; Mix extraction vortex 1min with equal-volume cyclohexane and distilled water, get the cyclohexane layer, add 12.5 μ L 1M HCl by every gram sample, vortex 1min leaves standstill 30min; Add the distilled water of 2 times of HCl volumes, 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH7.0～8.0 with 1N NaOH, after 10 times of dilutions of dilution, gets 80 μ L and analyzes.
(E) detection of heavy metal cadmium ion
(1) every micropore adds 10 μ L100mM EDTA solution respectively, and then adds the cadmium ion titer of 80 μ L series concentration (0,10,50,125,250,500,2000,5000ng/mL) and the above-mentioned flesh of fish sample extracting solution of handling well of 80 μ L respectively in micropore;
(2) adding 10 μ L, mix to having titer and oppressing in the micropore of sample extracting solution gently with the monoclonal antibody of the Cadmium resistance ion of dilution dilution in 1: 32000, hatch 2h for 37 ℃;
(3) wash micropore 3～5 times with cleansing solution;
(4) each micropore adds the goat anti-mouse igg antibody of 100 μ L with the HRP mark of dilution dilution in 1: 4000, hatches 1h for 37 ℃;
(5) wash micropore 3～5 times with cleansing solution;
(6) every micropore add 100 μ L colour developing liquid TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid, reaction 10～20min;
(7) every micropore adds 50 μ L 2M H
2SO
4(stop buffer) cessation reaction, and the mixing that vibrates gently;
(8) in 15 minutes, be determined at OD (absorbance) value under the 450nm wavelength with enzyme connection readout instrument.The titer reading is followed successively by 1.785,1.645,1.431,1.147,0.958,0.663,0.362,0.120, and the sample reading is 1.250.
(F) criterion as a result
Gained titer and sample absorbance be multiply by 100% again divided by the absorbance of 0 standard (titer of 0 concentration), are ordinate with this titer calculated value, and the logarithm of concentration of cadmium ions (μ g/kg) is a horizontal ordinate drawing standard curve.
B/B0 value according to each sample just can be read corresponding concentration of cadmium ions 92.826ppb from above-mentioned typical curve, multiply by corresponding extension rate again, and finally calculating concentration of cadmium ions actual in the sample is 928.26 μ g/kg.
Embodiment 2
Utilize Cadmium resistance ion monoclonal antibody to detect the indirect competitive ELISA method of cadmium ion, comprise the steps:
(A) with iEDTA cadmium ion is coupled on the hemocyanin (KLH), mix not formula Freund immunity BALB/c mouse, getting immune mouse spleen cell and SP2/0 myeloma cell merges, filter out the positive cell strain and the enlarged culture of stably excreting Cadmium resistance ion monoclonal antibody (MAb-Cd), the injection cell advances in the mouse body to induce ascites, and purifying obtains the monoclonal antibody of Cadmium resistance ion.
(B) with iEDTA cadmium ion is coupled to carrier protein BSA and goes up acquisition detection antigens c d-iEDTA-BSA.
(C) antigen is pre-wraps by the preparation of bar:
(1) bag quilt: (the 0.05M sodium carbonate buffer pH9.6) is diluted to Cd-iEDTA-BSA 0.2 μ g/mL, and every micropore 100 μ L join in the micropore of polystyrene micropore plate, and 4 ℃ of bags are spent the night to be cushioned liquid with bag.
(2) washing: after pouring out in the micropore plate hole bag and being cushioned liquid, microwell plate is upside down in pats on the thieving paper to guarantee to remove fully the liquid in the micropore.With the hyperchannel pipettor 200 μ l cleansing solutions are added in the micropore.Pour out the liquid in the micropore after 5 minutes once more, remove the liquid in the micropore fully.
(3) sealing: every micropore add 150 μ L contain 3% (W/V) BSA (Bovine serum album, bovine serum albumin(BSA)) 0.01M PBS (Phosphate-Buffered Saline, phosphate buffer, pH7.4), 37 ℃ the sealing 1h;
(4) set by step (2) wash micropore 5 times with cleansing solution;
(5) add 20% (W/V) sucrose phosphate buffer room temperature protection 3 hours;
(6) drying: after putting the hothouse drying, obtain antigen and wrap by bar in advance, preserve in the packaging bag that contains drying agent of packing into.
(D) sample pretreatment (shellfish meat sample extracting solution)
Get the shellfish meat sample that shreds and place centrifuge tube, add the 2mL ethyl acetate, fully smash even matter by every gram sample; The centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; Mix extraction vortex 1min with equal-volume cyclohexane and distilled water, get the cyclohexane layer, add 12.5 μ L 1M HCl by every gram sample, vortex 1min leaves standstill 30min; Add the distilled water of 2 times of HCl volumes, 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH7.0～8.0 with 1N NaOH, after 10 times of dilutions of dilution, gets 80 μ L and analyzes.
(E) detection of heavy metal cadmium ion
(1) every micropore adds 10 μ L100mM EDTA solution respectively, and then adds the cadmium ion titer of 80 μ L series concentration and the above-mentioned shellfish meat sample extracting solution of handling well of 80 μ L respectively in micropore;
(2) add 10 μ L1: the monoclonal antibody of the Cadmium resistance ion of 32000 dilutions is mixed in the micropore of existing titer and shellfish meat sample extracting solution gently, hatches 0.5h for 37 ℃;
(3) wash micropore 3～5 times with cleansing solution;
(4) each micropore adds 100 μ L1: the goat anti-mouse igg antibody of the HRP mark of 4000 dilutions, hatch 0.5h for 37 ℃;
(5) wash micropore 3～5 times with cleansing solution;
(6) every micropore add 100 μ L colour developing liquid TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid, reaction 10～20min;
(7) every micropore adds 50 μ L 2M H
2SO
4(stop buffer) cessation reaction, and the mixing that vibrates gently;
(8) in 15 minutes, be determined at OD (absorbance) value under the 450nm wavelength with enzyme connection readout instrument.The titer reading is followed successively by 1.757,1.684,1.419,1.128,0.955,0.659,0.344,0.126, and the sample reading is 1.404.
(F) criterion as a result
Gained titer and sample absorbance be multiply by 100% again divided by the absorbance of 0 standard (titer of 0 concentration), are ordinate with this titer calculated value, and the logarithm of concentration of cadmium ions (μ g/kg) is a horizontal ordinate drawing standard curve.
B/B0 value according to each sample just can be read corresponding concentration of cadmium ions 58.288ppb from above-mentioned typical curve, multiply by corresponding extension rate again, finally calculates concentration of cadmium ions 582.88 μ g/kg actual in the sample.
For checking effect of the present invention gets reliability, carry out following evaluation:
Get the ELISA Plate of having sealed, every micropore adds 10 μ L 100mM EDTA solution, adds the Cd that 80 μ L prepare
2+Standard solution (0,10,50,125,250,500,2000,5000ng/mL) mixing is as competition antigen, the monoclonal antibody working fluid (1: 32000 times) that adds the best dilution of 10 μ L again, mixing adds 100 μ L cleansing solutions as the blank hole that does not add monoclonal antibody in a hole; 37 ℃ of incubation 1.5h wash plate) add with the HRP enzyme mark goat-anti mouse two of dilution dilution in 1: 4000 anti-ly, 37 ℃ of incubation 1h add 100 μ L tmb substrate liquid colour developing 10 minutes, with 50 μ L2M H
2SO
4Stop, with microplate reader interpretation under the 450nm wavelength.With Cd
2+The concentration logarithm is a horizontal ordinate, B/B
0(%) be ordinate, drawing standard suppresses curve.
Can see (B/B in the 0.050-2 μ g/ml scope from Fig. 1, Fig. 2
0) and Cd
2+The logarithm value of concentration is the good linear relation, and related coefficient is r=0.9978, Cd during with 10% inhibiting rate
2+Concentration is lowest detectable limit, reaches 0.010 μ g/ml.
(2) repeatability as a result
At Cd
2+The precision test that detects repeats 5 times between batch, and the light absorption value coefficient of variation (CV%) of gained variable concentrations standard solution is as shown in table 1, shows that the inventive method has very high repeatability.
The interassay coefficient of variation of table 1 competitive ELISA
| Cd 2+(ng/mL) | 10 | 50 | 125 | 250 | 500 | 1000 | 2000 |
| The coefficient of variation (%) | 2.32 | 3.83 | 1.44 | 3.42 | 4.00 | 2.89 | 7.72 |
(3) recovery is calculated
Get in the blank flesh of fish of the 10g sample and add Cd
2+Standard specimen after the extract dilution (being diluted to about within the concentration range of typical curve), carries out ELISA and measures.Check in Cd according to OD value and B/B0 value from typical curve
2+Content multiply by extension rate again and is Cd in the sample
2+Content, calculate recovery rate sees Table 2 then.
The recovery of table 2 working sample
| Add the amount (μ g/g) of Cd2+ in the sample | The amount of the Cd2+ that measures | The |
| 50 | 50.56±1.4 | 101.12±2.8 |
| 5 | 4.81±0.2 | 96.34±4.0 |
| 0.5 | 0.46±0.01 | 93.17±2.0 |
The recovery 93～101% as can be seen from the table.
(4) specificity as a result
Carry out specificity cross reaction test at following heavy metal utilization competitive ELISA, the result is as follows:
Other several cross reactions of table 3 heavy metal cadmium
| Metallic ion | IC 50(ng/mL) | Cross reacting rate (%) |
| Cd 2+ Hg 2+ Cu 2+ Mn 2+ Zn 2+ Fe 3+ Ca 2+ | 250 200 ＞100000 ＞100000 ＞300000 ＞500000 ＞500000 | 100 125 ＜0.25 ＜0.25 ＜0.08 ＜0.05 ＜0.05 |
| pb 2+ Ni 2+ Al 3+ Mg 2+ | ＞5000000 ＞5000000 ＞5000000 ＞5000000 | ＜0.005 ＜0.005 ＜0.005 ＜0.005 |
The result shows that antibody removes Cd
2+In addition, to Hg
2+Tangible intersection is arranged, and then cross reaction is very little to other several metals, and therefore, this monoclonal antibody can be used for detecting simultaneously Cd
2+And Hg
2+Two heavy metal species have enlarged range of application.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the detection method of a cadmium ion indirect competitive ELISA, it is characterized in that comprising the steps: that respectively cadmium ion titer with fish and shellfish sample extracting solution and series concentration joins antigen and wraps in advance by in the micropore of bar, the monoclonal antibody that adds the Cadmium resistance ion again is to each micropore, mixing is hatched, in each micropore, add the goat anti-mouse igg antibody of HRP mark, hatch; Cessation reaction after substrate colour developing is measured the absorbance of each micropore, the Regression Equations of reference standards gained, the content of cadmium ion in the calculation sample with microplate reader.
2. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 1 is characterized in that comprising the steps:
(1) in antigen wraps by each micropore of bar in advance, all adds the 100mM EDTA of 10 μ l respectively, in above-mentioned each micropore, add the Cd of 80 μ l fish and shellfish sample extracting solutions and 80 μ L series concentration then more respectively
2+Titer, mixing; Each micropore adds the monoclonal antibody of the Cadmium resistance ion of 1: 32000 times of dilution of 10 μ L then; Mixing is hatched 0.5～2h for 37 ℃; Wash micropore with cleansing solution then;
(2) each micropore adds the goat anti-mouse igg antibody of 100 μ L with the HRP mark of dilution dilution in 1: 4000, hatches 0.5～1h for 37 ℃; Wash micropore with cleansing solution then;
(3) each micropore adds 100 μ L colour developing liquid tmb substrate working fluid, reaction 10～20min;
(4) each micropore adds 50 μ L stop buffer 2M H
2SO
4Cessation reaction, and vibration mixing;
(5) be determined at absorbance under the 450nm wavelength with enzyme connection readout instrument;
(6) gained titer and sample absorbance being multiply by 100% again divided by the absorbance of 0 standard, is ordinate with this titer calculated value, and the logarithm of concentration of cadmium ions is a horizontal ordinate drawing standard curve; B/B according to each sample
0Value is read corresponding concentration of cadmium ions from typical curve, multiply by corresponding extension rate again, calculates concentration of cadmium ions actual in the sample; Described dilution and cleansing solution are the 0.01M phosphate buffer that PBS-T promptly contains 0.05% Tween-20.
3. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2 is characterized in that: the pre-service as follows of described fish and shellfish sample extracting solution: every gram flesh of fish or shellfish meat sample add the 2mL ethyl acetate, fully smash even matter; The centrifugal 10min of 4000rpm/min gets supernatant nitrogen stream under 50 ℃ of water-baths and dries up sample; Mix extraction vortex 1min with equal-volume cyclohexane and distilled water, get the cyclohexane layer, every gram sample adds 12.5 μ L 1M HCl, and vortex 1min leaves standstill 30min again; Add the distilled water of 2 times of HCl volumes, 8000rpm/min discards the upper strata cyclohexane, draws subnatant, adjusts to pH7.0～8.0 with 1N NaOH, after 10 times of diluted, promptly gets the fish and shellfish sample extracting solution.
4. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2 is characterized in that: the pre-bag of described antigen is prepared as follows by bar:
(1) bag quilt: to be cushioned liquid be that the 0.05M sodium carbonate buffer of pH9.6 is diluted to 0.2 μ g/mL with Cd-iEDTA-BSA with wrapping, and every micropore 100 μ L join in the polystyrene micropore plate hole, 4 ℃ of bags spent the night or 37 ℃ of bags by 2～3h; Described Cd-iEDTA-BSA is coupled to carrier protein BSA with iEDTA with cadmium ion and goes up acquisition;
(2) washing: after pouring out in the micropore plate hole bag and being cushioned liquid, 200 μ l cleansing solutions are added in each micropore; Pour out the liquid in the hole after 3～5 minutes once more, remove the liquid in the micropore fully; Described cleansing solution is the 0.01M phosphate buffer that contains 0.05% Tween-20;
(3) sealing: add the 0.01MPBS that 150～200 μ L contain 0.5%～3%BSA at the every micropore of microwell plate, 37 ℃ of sealing 0.5h～1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% sucrose phosphate buffer room temperature protection 3 hours;
(6) drying: after the microwell plate drying, promptly obtain antigen and wrap by bar in advance.
5. the detection method of a kind of cadmium ion indirect competitive ELISA according to claim 2, the monoclonal antibody that it is characterized in that described Cadmium resistance ion is prepared from as follows: with iEDTA cadmium ion is coupled on the hemocyanin, mix not formula Freund immunity BALB/c mouse, getting immune mouse spleen cell and SP2/0 myeloma cell merges, filter out the positive cell strain and the enlarged culture of stably excreting Cadmium resistance ion monoclonal antibody, the injection cell advances in the mouse body to induce ascites, and purifying obtains the monoclonal antibody of Cadmium resistance ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100329146A CN101196521A (en) | 2007-12-27 | 2007-12-27 | Indirectly racing ELISA detecting method for cadmium ion |
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| CN101968481A (en) * | 2010-08-13 | 2011-02-09 | 中国农业大学 | Method for detecting existence of heavy metal copper ion in sample and special kit thereof |
| CN102539742A (en) * | 2011-12-13 | 2012-07-04 | 暨南大学 | Method for detecting heavy metals in carbohydrate agricultural products and application thereof |
| CN104007262A (en) * | 2014-05-16 | 2014-08-27 | 中南林业科技大学 | Cadmium ion colloidal gold immunochromatographic assay test paper strip and preparation method and application thereof |
| CN104155424A (en) * | 2014-08-19 | 2014-11-19 | 河南华牧生物科技有限公司 | Method for detecting cadmium ion pollution in water by blocking ELISA (Enzyme-Linked Immunosorbent Assay) method |
| CN104749364A (en) * | 2013-12-25 | 2015-07-01 | 深圳先进技术研究院 | Immune analysis method and immune analysis kit for detecting zinc |
| CN104762268A (en) * | 2015-03-11 | 2015-07-08 | 暨南大学 | High-affinity heavy-metal-cadmium-resistant monoclonal antibody and application thereof |
| CN104987407A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Cadmium-IgG chelate as well as preparation method and application thereof |
| CN105622758A (en) * | 2014-10-29 | 2016-06-01 | 中国科学院上海生命科学研究院 | Nanobody for specific recognition of heavy metal Cd and application thereof |
| CN108362873A (en) * | 2018-02-13 | 2018-08-03 | 苏州仁端生物医药科技有限公司 | A kind of cadmium ion detection kit and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101968481B (en) * | 2010-08-13 | 2013-12-11 | 中国农业大学 | Method for detecting existence of heavy metal copper ion in sample and special kit thereof |
| CN101968481A (en) * | 2010-08-13 | 2011-02-09 | 中国农业大学 | Method for detecting existence of heavy metal copper ion in sample and special kit thereof |
| CN102539742B (en) * | 2011-12-13 | 2014-12-03 | 暨南大学 | Method for detecting heavy metals in carbohydrate agricultural products and application thereof |
| CN102539742A (en) * | 2011-12-13 | 2012-07-04 | 暨南大学 | Method for detecting heavy metals in carbohydrate agricultural products and application thereof |
| CN104749364A (en) * | 2013-12-25 | 2015-07-01 | 深圳先进技术研究院 | Immune analysis method and immune analysis kit for detecting zinc |
| CN104007262A (en) * | 2014-05-16 | 2014-08-27 | 中南林业科技大学 | Cadmium ion colloidal gold immunochromatographic assay test paper strip and preparation method and application thereof |
| CN104155424A (en) * | 2014-08-19 | 2014-11-19 | 河南华牧生物科技有限公司 | Method for detecting cadmium ion pollution in water by blocking ELISA (Enzyme-Linked Immunosorbent Assay) method |
| CN105622758A (en) * | 2014-10-29 | 2016-06-01 | 中国科学院上海生命科学研究院 | Nanobody for specific recognition of heavy metal Cd and application thereof |
| CN104762268A (en) * | 2015-03-11 | 2015-07-08 | 暨南大学 | High-affinity heavy-metal-cadmium-resistant monoclonal antibody and application thereof |
| CN104762268B (en) * | 2015-03-11 | 2017-12-29 | 暨南大学 | A kind of high-affinity preventing from heavy metal cadmium monoclonal antibody and its application |
| CN104987407A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Cadmium-IgG chelate as well as preparation method and application thereof |
| CN104987407B (en) * | 2015-07-14 | 2018-01-05 | 上海拜豪生物科技有限公司 | cadmium-IgG chelate as well as preparation method and application thereof |
| CN108362873A (en) * | 2018-02-13 | 2018-08-03 | 苏州仁端生物医药科技有限公司 | A kind of cadmium ion detection kit and its application |
| CN108535467A (en) * | 2018-03-26 | 2018-09-14 | 暨南大学 | A kind of kit of chemiluminescence indirect competition immune detection copper ion and its application |
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