CN101655499A - Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury - Google Patents
Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury Download PDFInfo
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Abstract
The invention discloses an indirect competitive enzyme-linked immunosorbent assay (ELISA) for measuring heavy metal mercury, belonging to a method for measuring heavy metal mercury in an environment water sample. The method comprises the following steps: taking a monoclonal antibody for specially identifying mercury-chelating agent EDTA compound Hg-EDTA as the base, coating the coated antigen mercury-chelating agent-albumen on a 96-hole ELISA plate; incubating at 4 DEG C over night and then sealing by phosphate buffer solution PBS including 1% of gelatin, after washing the ELISA plate, addinga mixing solution of the specificity mercury monoclonal antibody and samples to be measured, incubating at 37 DEG C and then washing the plate, adding an ELISA secondary antibody, incubating at 37 DEGC, adding an enzyme reaction substrate after washing the plate, and adding a reaction stop solution for stopping the reaction after the incubation, and then measuring the absorbance value of each hole by an ELISA reader; obtaining a standard competitive inhibit curve, then executing Logit transition on the curve, and drawing the standard curve of the mercury in the sample to carry out the quantitative analysis. The method is suitable for the measurement of trace mercury in an environment water sample, simultaneously provides the technical support for the fast scene measurement of heavy metalpollution emergency in order to remedy the pollution accident and go back to work in time.
Description
Technical field
The present invention relates to a kind of quick, accurate, high throughput assay method of heavy metal Hg, be specially a kind of indirect competitive enzyme-linked immunosorbent assay for measuring (ELISA method) based on heavy metal mercury monoclonal antibody.
Background technology
Heavy metal generally extensively is present in occurring in nature with natural concentration, and mercury content is few in the natural water body, generally is no more than 0.1 μ g/L, and the Drinking Water in China standard limited value is 0.001mg/L.But it is because human increasing to exploitation, smelting, processing and the commercial manufacturing activities of heavy metal, cause many heavy metals such as cadmium, mercury etc. to enter atmosphere, water, soil environment, cause the serious environmental pollution, the heavy metal that exists with various chemical states or chemical form, after entering the environment or the ecosystem, will retain, accumulate and move, work the mischief, and can be by potable water or the health by final harm humans of mode such as biological concentration and food chains.Therefore, set up detection method accurate, quick, convenient, high-throughout heavy metal Hg, mercury pollution is carried out long term monitoring, understand its concentration in surrounding medium, control is polluted, protection environment and human life's safety and health have crucial meaning.
Literature search is the result show, external Willy etc. has prepared the monoclonal antibody (Wylie of energy specific recognition heavy metal Hg first, D.E., et al.Monoclonal-antibodies specific for mercuric ions[J] .Proceedings of the National Academy of Sciences of the United States ofAmerica, 1992,89 (9): 4104-4108), and tentatively be used for the detection of water body mercury, but sensing range is relatively narrow, be 0.5-10ppb (Wylie, D.E., et al.Detection of mercuric ions inwater by Elisa with a mercury-specific antibody[J] .Analyticai Biochemistry, 1991,194 (2): 381-387).The beautiful grade of domestic Yang Feng has been delivered on " hi-tech communication " (2008.5) and has been entitled as " preparation of heavy metal mercury monoclonal antibody and evaluation ", and this article has been introduced the method and the antibody that prepare mercury monoclonal antibody and identified.But all being not used in, these set up standardized immunologic detection method.
Current, in the world the developing direction of counterweight metal detection be sensitive, accurately, in real time, fast, selectivity is good and applied widely etc.And the detection technique of traditional mercury needs large-scale mostly or specialized equipment (atomic absorption spectrography (AAS), atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry method etc.) just can be finished, and pre-treatment process complexity, consuming time more, analytical work can only be carried out indoor, is difficult to satisfy the needs of the quick and online detection of high flux.Therefore, develop the new method and the new technology that are used for the fast detecting heavy metal Hg and have realistic meaning.But, slow about the progress of this respect both at home and abroad at present, do not form effective mature technology as yet.Based on the indirect competitive enzyme-linked immunosorbent assay for measuring of antigen, antibody specificity combination have that detection speed is fast, expense is cheap, advantage such as the simple portable of instrument, highly sensitive and selectivity are strong, and can analyze a large amount of environmental samples simultaneously, be widely used in the detection of organic toxic substance in clinical medicine, life science and the environment.But in the detection that is used for heavy metal Hg, do not have breakthrough always.
Summary of the invention
1. invent the technical matters that will solve:
Problem at existing heavy metal cadmium mercury detection method existence, the invention provides a kind of indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury, based on quick, accurate, the high-throughout indirect competitive enzyme-linked immunosorbent determining adsorption of the mercury monoclonal antibody that high specific, height are tired, can be used for the fast detecting of environmental water sample.
2. technical scheme
The present invention is achieved through the following technical solutions:
A kind of indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury (being indirect competitive enzyme-linked immunosorbent assay for measuring (CI-ELISA)), monoclonal antibody based on specific recognition mercury-sequestrant EDTA compound Hg-EDTA, at first coating antigen mercury-sequestrant-albumen (Hg-ITCBE-BSA) is coated on the 96 hole ELISA Plate, seal with the phosphate buffer PBS that contains 1% gelatin after 4 ℃ of overnight incubation, wash the mixed solution that adds specificity mercury monoclonal antibody and testing sample behind the plate, wash version after hatching for 37 ℃, add ELIAS secondary antibody, hatch for 37 ℃, add enzyme reaction substrate after washing plate, hatch the back and add cessation reaction liquid cessation reaction, measure each hole absorbance with microplate reader then.
Below by detailed steps the present invention is done further concrete the qualification:
In advance 1000mg/L mercury metal titer is diluted to the 200mg/L storing solution, regulates pH to desired value.Dispose certain density EDTA solution with dilution buffer liquid PBS, be PBSE solution.Storing solution is diluted to the standard solution of series concentration with distilled water.The Hg-EDTA complex solution that reaction generates series concentration with EDTA adds ELISA Plate as competing compound again.In this process, the amount that guarantees to contain in the mark liquid of each concentration EDTA is consistent.Concrete experimental procedure is as follows:
(1) bag quilt: be cushioned solution C BS dilution coating antigen to the antigen working concentration with bag, 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours.
(2) wash plate: be washing lotion with PBST, wash washing on the plate machine that every plate is given a baby a bath on the third day after its birth time, each every hole fluid injection 300 μ L pat dry.
(3) sealing: with 1% gelatin is confining liquid, and 1h is hatched for 37 ℃ in 200 μ L/ holes.
(4) wash plate; With (2), pat dry.
(5) premixed: the Hg that gets series concentration
2+Each 100 μ L of solution are with after 900 μ LEDTA mix, and the antibody equal-volume after mixed liquor and PBS are diluted is mixed to 1mL, at room temperature acts on a period of time.Do positive control with EDTA with dilution antibody mixed liquor simultaneously.
(6) competition: the standard model of each concentration that will be pre-mixed adds in the 96 hole ELISA Plate, and 2h is hatched for 37 ℃ in 100 μ L/ holes.
(7) wash plate:, pat dry with (2).
(8) add sheep anti mouse ELIAS secondary antibody: sheep anti-mouse igg-HRP with confining liquid dilution (1: 10000), 1h is hatched for 37 ℃ in 100 μ L/ holes.
(9) wash plate:, pat dry with (2).
(10) add substrate solution (TMB of 100 μ L 10mg/mL and 25 μ L 0.65%H
2O
2Be dissolved in 9.875mL CPBS, now join use before the use): 100 μ L/ holes, 37 ℃ of colour developing 15min.
(11) cessation reaction: adding concentration is the sulfuric acid solution color development stopping reaction of 2M, 50 μ L/ holes.
(12) measure, calculate: microplate reader is measured OD
450Value, with blank zeroing, with EDTA and antibody mixed liquor hole as positive control.The OD that positive control hole (not containing competing compound) and well (containing the finite concentration competing compound) record
450Difference and the OD that records of positive control hole
450Ratio be inhibiting rate.Computing formula is as follows:
By logarithm value mapping to the concentration of mercury in inhibiting rate and the standard model, obtain the standard competition and suppress curve, again curve is carried out the Logit conversion, promptly the Logit value with each hole absorbance is an ordinate, negative logarithm with the concentration of mercury in the standard model is a horizontal ordinate, draw out the typical curve of mercury in the sample, in the Logit value of each hole absorbance and the standard model negative logarithm of the concentration of mercury be in line relevant, thereby carry out quantitative test.
Described mercury monoclonal antibody; be to be immunogene through Hg-ITCBE-KLH; take the mode immunity BALB/c mouse of lumbar injection; and obtain through hybridoma technology; it is not the claimed inventive point of the present invention herein; can oneself prepare, also can provide to obtain by the preparation side of mercury monoclonal antibody.
Ultimate principle of the present invention is at first coating antigen to be coated on the ELISA Plate, then antibody and micromolecule haptens potpourri is added micromolecule haptens and be coated on competitive association reaction takes place between the envelope antigen on the ELISA Plate as sample.The haptenic concentration of micromolecule is high more in the sample, and the amount of the antibody of combination is relatively just many more with it, and the antibody amount that is combined on the envelope antigen is just few more, and after the adding ELIAS secondary antibody, the chromogenic reaction of generation is just shallow more, OD
450Be worth low more.Vice versa.Pass through OD
450Sxemiquantitative or quantitative test are carried out in the variation of value.
3. beneficial effect:
Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury provided by the invention, make that heavy metal Hg is quick, accurate, high throughput assay, on the basis of high specific mercury monoclonal antibody, set up the indirect competitive enzyme-linked immunosorbent assay for measuring of measuring mercury content in the environmental water sample.This method is fit to Determination of Trace Hg in the environmental water sample.Field quick detection for the heavy metal pollution burst accident provides technical support simultaneously, be convenient in time contamination accident be remedied and resumed work, and can develop into the important detection means of other samples and agricultural product food safety detection in the environment, be of great immediate significance.
Description of drawings
Fig. 1 indirect competitive enzyme-linked immunosorbent assay for measuring standard suppresses curve
The quantitative curve of Fig. 2 indirect competitive enzyme-linked immunosorbent assay for measuring Logit
Embodiment
Below further specify the present invention by example.
Embodiment 1: be used to detect the preparation of heavy metal mercury monoclonal antibody
Adopt isothiocyanates method synthesizing heavy metal mercury comlete antigen, combine by the amino that dissociates on the thiocyanate on the bifunctional chelating agent in the haptens compound and the macromolecular carrier albumen, thereby generated comlete antigen.Be specially: with isothiocyano-benzyl-ethylenediamine tetraacetic acid (ITCBE) as the bifunctional chelating agent agent, form mercury-sequestrant (Hg-ITCBE) compound with heavy metal cadmium, again respectively with bovine serum albumin(BSA) (BSA) and keyhole relative hemocyanin (KLH) as carrier protein and the coupling of Hg-ITCBE compound, final Hg-ITCBE-BSA and Hg-ITCBE-KLH comlete antigen, i.e. coating antigen and the immunogene of forming.
Get the female BALB/c mouse in 46 ages in week, in immunity docking the last week blood sampling, as negative serum.After one week, be that immunogene is carried out immunity, take the mode of lumbar injection to carry out five immunity altogether with Hg-ITCBE-KLH.Each immunizing antigen consumption is 200 μ g/ only (in protein concentrations), initial immunity with immunizing antigen with the emulsification of equivalent Freund's complete adjuvant, two, three, four, five immunity and the emulsification of equivalent incomplete Freund, each immunity 2 weeks of interval.Merged preceding 3 days, with the direct booster immunization of antigen once, dosage is the same.Third and fourth, five immunity backs adopted the docking modes to gather serum on the 10th day, survey antibody titer by indirect non-competing ELISA method, with Hg-ITCBE-BSA and ITCBE-BSA respectively as the coating antigen coated elisa plate, relatively mouse resisting anteserum is measured antiserum and is tired with combining of they affinity of two kinds of coating antigens.Select higher and the two OD of antibody titer at last
450The mouse that differs greatly is used for Fusion of Cells.
Tire higher and the two OD with the hybridoma technology antagonist
450Splenocyte and the sp2/0 myeloma cell of the mouse that differs greatly are merged, and obtain hybridoma, and hybridoma is screened, and obtain the energy stably excreting has the specific recognition function to Hg-EDTA monoclonal antibody.
Get 10 age in week Healthy female Balb/C mouse, with the 0.5mL whiteruss every mouse is carried out lumbar injection, make mouse produce sensitizing effect, after 7～14 days, every mouse peritoneal injection 1～2 * 10
6Individual hybridoma is observed the mouse state, after 7～10 days, treats that mouse peritoneal obviously expands the aseptic collection ascites in back, the centrifugal 15min of 4000rpm, and supernatant is ascites, and packing, mark are standby in-20 ℃ of preservations; 2～3 days at interval, treat that ascites regeneration is gathered after, get again with method, a general mouse can extract 2～3 times.Treat that mouse ascites all takes out back purifying ascites, indirect non-competing ELISA method is surveyed it and is tired.Ascites is tired and is reached 1: 300000.
Embodiment 2: the specific evaluation of mercury monoclonal antibody
Present embodiment has been selected in the environment some common heavy metal ion to carry out cross reacting rate to measure, investigate the specificity of mercury monoclonal antibody.The metallic ion of selecting has: Zn
2+, Pb
2+, Cd
2+, Al
3+, Ni
2+, Mg
2+, Ca
2+, Co
2+, Cu
2+Deng 9 kinds.Concrete experimental procedure according to summary of the invention indirect competitive enzyme-linked immune determining adsorption method is operated.With concentration is 0.1,1,10,20,50,100,500,1000 μ g/LHg
2+Standard solution and concentration are 0.01,0.1,1,5,10, the Zn of 50mg/L
2+, Pb
2+, Cd
2+, Al
3+, Ni
2+, Mg
2+, Ca
2+, Co
2+, Cu
2+Series standard solution adds in the ELISA Plate as sample, the OD in each hole in the assay plate
450Value.According to suppress curve produce 50% suppress or combination respectively compete substrate concentration IC
50(mg/L), the compatibility that compares their antagonists.Calculate the cross reacting rate (Cross-Reactivity) of other competition things with following formula simultaneously, to investigate the specificity of antibody to Hg.
Cross reacting rate CR%=[IC
50(CP)/IC
50(competitor)] * 100%
The height of cross reacting rate has determined them to Hg
2+Detect the size of annoyance level, promptly method is to the specificity of Hg.Data result is listed in table 1.As can be seen from the table, the maximal percentage inhibition of other 9 kinds of metal pair antibody all is lower than 50%, and cross reacting rate is all less than 0.13%.Experimental result shows that the monoclonal antibody of the present invention's preparation has good specificity to mercury, and it is more weak to his affinity of metallic ion.Therefore, when using monoclonal antibody of the present invention to carry out the enzyme linked immunosorbent assay (ELISA) analysis, whether there is heavy metal Hg very accurately in the test sample, thereby carries out quantitative test.
Embodiment 3: the heavy metal Hg enzyme-linked immunosorbent assay for measuring
1: the determining of antigen-antibody best effort concentration
Adopt the square formation titrimetry that the best effort concentration of envelope antigen and antibody is optimized: is the 1-3 of 1000 times, 2000 times, 4000 times, 8000 times coated elisa plates with CBS with envelope antigen Hg (II)-ITCBE-BSA serial dilution, 4-5,6-9,10-12 row; With 1% gelatin as confining liquid; Antibody doubling dilution respectively is 5000 times, 10000 times, 20000 times, 40000 times, 80000 times, 160000 times, 320000 times and 640000 times, and is capable in the A-H of ELISA Plate; Add sheep anti mouse two anti-IgG-HRP (1: 5000, the PBS dilution); The TMB colour developing, 2M sulfuric acid stops, and surveys OD
450Value.With OD
450Value for be slightly larger than level off to 1 and antigen and antibody concentration be best effort concentration when being minimum combination.Experimental result shows that the antigen diluent degree is 4000-8000, and antibody dilution is 80000 o'clock, OD
450Value is 1.092-1.247.This experiment with antigen diluent degree 5000 as best antigen diluent concentration.
The foundation of 2:ELISA method
According to the concrete qualification step of summary of the invention indirect competitive enzyme-linked immune determining adsorption method, set up the assay method of mercury:
(1) bag quilt: be cushioned solution C BS dilution coating antigen to the antigen working concentration with bag, 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours;
(2) wash plate: be washing lotion with PBST, wash washing on the plate machine that every plate is given a baby a bath on the third day after its birth time, each every hole fluid injection 300 μ L pat dry;
(3) sealing: with 1% gelatin is confining liquid, and 1h is hatched for 37 ℃ in 200 μ L/ holes.
(4) wash plate; Same step (2) pats dry;
(5) premixed: the Hg that gets series concentration
2+Each 100 μ L of solution are with after 900 μ LEDTA mix, and the antibody equal-volume after mixed liquor and PBS are diluted is mixed to 1mL, at room temperature acts on a period of time.Do positive control with EDTA with dilution antibody mixed liquor simultaneously;
(6) application of sample reaction: the standard model of each concentration that will be pre-mixed adds in the 96 hole ELISA Plate, and 2h is hatched for 37 ℃ in 100 μ L/ holes;
(7) wash plate:, pat dry with (2);
(8) add sheep anti mouse ELIAS secondary antibody: sheep anti-mouse igg-HRP with confining liquid dilution (1: 10000), 1h is hatched for 37 ℃ in 100 μ L/ holes;
(9) wash plate:, pat dry with (2);
(10) add substrate solution (TMB of 100 μ L 10mg/mL and 25 μ L 0.65%H
2O
2Be dissolved in 9.875mL CPBS, now join use before the use): 100 μ L/ holes, 37 ℃ of colour developing 15min;
(11) cessation reaction: adding concentration is the sulfuric acid solution color development stopping reaction of 2M, 50 μ L/ holes;
(12) measure, calculate: microplate reader is measured OD
450Value, with blank zeroing, with EDTA and antibody mixed liquor hole as positive control, the OD that positive control hole and well record
450Difference and the OD that records of positive control hole
450Ratio be inhibiting rate, computing formula is as follows:
3: condition optimizing
On best elisa assay method working concentration basis,, select blank OD with the coating buffer bag quilt of different pH values and ionic strength
450Be worth higher relatively bag by condition as the best.The present invention adopts the CBS of pH=9.6 to be cushioned liquid as bag.
Investigate of the influence of different salt ionic concentrations, make that the concentration of NaCl changes successively in the reaction system, select blank OD antigen-antibody binding reaction
450Be worth higher relatively salt ionic concentration.The present invention adopts the solution of 0.15M as dilution buffer liquid.
The effect of confining liquid is to eliminate non-specific adsorption.In the competitive ELISA reaction, under the condition of antigen-antibody best effort concentration and the suitableeest salt ion intensity, the present invention has adopted 1% gelatin, 3%BSA, 1%OVA respectively, has reached 5% skimmed milk power as confining liquid (200 μ L/ hole), relatively its influence to detection curve.The present invention adopts 1% gelatin as confining liquid according to experimental result.
Change EDTA concentration in the metal mark liquid dilution buffer liquid, make that EDTA concentration is respectively 2.5mM in the reaction system, 5.0mM, 10mM, 20mM, the inhibiting effect of more different EDTA concentration antagonists.According to the result, the present invention adopts the concentration of 5.0mM EDTA as EDTA in the sample buffer.
The pH value is got 3-4,7.4,7.8 respectively in the reaction system, and more different pH values are to the influence of ELISA detection curve.According to experimental result, reaction environment pH does not have evident difference between 7 and 7.8, and it is 7.4 that the present invention adopts pH value of reaction system.
4: typical curve
Under above-mentioned optimum reaction condition, set up indirect competitive enzyme-linked immunosorbent assay for measuring, be ordinate (Y) with inhibiting rate Inhibition rate, Hg
2+The negative logarithm of concentration (μ g/L) is that the competition of horizontal ordinate (X) drawing standard suppresses curve, as shown in Figure 1.As can be seen from the figure Hg
2+In 0.1 μ g/L～1000 μ g/L scopes, inhibiting rate and Hg
2+The negative logarithm related coefficient of concentration μ g/L reaches 0.999, the sensing range broad of method.From figure, also can obtain IC
50Be 13.27 μ g/L, IC
20Be 0.468 μ g/L, with IC
20Concentration be lowest detectable limit, then this method is to Hg
2+The detection of concentration is limited to 0.459 μ g/L.The maximum permissible concentration of China's domestic water and farmland irrigation water is 0.001mg/L (1 μ g/L).As seen the indirect competitive ELISA method is applicable to the requirement of water quality detection fully.
5: quantitative Analysis and analysis
Standard inhibition curve in 4 is carried out the Logit conversion, promptly with Logit (B/B
0) be ordinate (Y), Hg
2+The negative logarithm of concentration (μ g/L) is horizontal ordinate (X), does standard Logit curve, and wherein B is the OD under the standard series concentration
450Value, B
0The OD of positive contrast
450Value.Logit (B/B
0) and Hg
2+The negative logarithm of concentration (μ g/L) is linear, as shown in Figure 2.Linear equation is Y=0.86837+0.22795X, R
2=0.994.
OD according to testing sample
450Be worth, calculate the Logit value of each sample, the linear equation of the above-mentioned typical curve of substitution is obtained the concentration of mercury in the corresponding sample of corresponding Logit value and is born logarithm, can calculate the content of mercury in the testing sample easily.
Embodiment 4: use this law the actual water sample mark-on is reclaimed mensuration
The TAIHU LAKE of get a certain amount of ultrapure water, tap water, handling after filtration under the situation of term harmonization, is added certain amount of H g respectively when being to set up the standard detection curve
2+Standard solution is used for the indirect competitive ELISA analysis behind serial dilution.According to OD
450Value and inhibiting rate value obtain corresponding Hg from typical curve
2+Concentration is added the recovery and is calculated as follows: adds recovery %=measured concentration/theoretical concentration * 100%.
Presentation of results, ultrapure water, tap water, the blank OD that obtains with the PBSE blank of TAIHU LAKE
450The value there was no significant difference, the interpret sample blank does not produce interference to antigen-antibody binding reaction.The interpolation recovery that adopts the ELISA method to measure ultrapure water, tap water, TAIHU LAKE is respectively 94.3%～123.5%, 91.85%～117.9%, 92.26%～115.6%; CV% is respectively 2.43%～6.57%, and 1.21%～4.79%, 1.12%～9.4%, meet the analysis requirement.
Table 1 antibody specificity experimental result
Claims (4)
1. an indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury is characterized in that based on the monoclonal antibody of specific recognition mercury-sequestrant EDTA compound Hg-EDTA coating antigen mercury-sequestrant-albumen being coated on the 96 hole ELISA Plate; Seal with the phosphate buffer PBS that contains 1% gelatin after 4 ℃ of overnight incubation, wash the mixed solution that adds specificity mercury monoclonal antibody and testing sample behind the plate, wash version after hatching for 37 ℃, add ELIAS secondary antibody, hatch for 37 ℃, add enzyme reaction substrate after washing plate, hatch the back and add cessation reaction liquid cessation reaction, measure each hole absorbance with microplate reader then.
2. according to claims 1 described indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury, it is characterized in that doing further concrete the qualification by following steps:
(1) bag quilt: be cushioned solution C BS dilution coating antigen to the antigen working concentration with bag, 100 μ L/ holes, 4 ℃ spend the night or 37 ℃ 2 hours;
(2) wash plate: be washing lotion with PBST, wash washing on the plate machine that every plate is given a baby a bath on the third day after its birth time, each every hole fluid injection 300 μ L pat dry;
(3) sealing: with 1% gelatin is confining liquid, and 1h is hatched for 37 ℃ in 200 μ L/ holes.
(4) wash plate; Same step (2) pats dry;
(5) premixed: the Hg that gets series concentration
2+Each 100 μ L of solution are with after 900 μ LEDTA mix, and the antibody equal-volume after mixed liquor and PBS are diluted is mixed to 1mL, at room temperature acts on a period of time.Do positive control with EDTA with dilution antibody mixed liquor simultaneously;
(6) application of sample reaction: the standard model of each concentration that will be pre-mixed adds in the 96 hole ELISA Plate, and 2h is hatched for 37 ℃ in 100 μ L/ holes;
(7) wash plate:, pat dry with (2);
(8) add sheep anti mouse ELIAS secondary antibody: sheep anti-mouse igg-HRP with confining liquid dilution (1: 10000), 1h is hatched for 37 ℃ in 100 μ L/ holes;
(9) wash plate:, pat dry with (2);
(10) add substrate solution (TMB of 100 μ L 10mg/mL and 25 μ L 0.65%H
2O
2Be dissolved in 9.875mLCPBS, now join use before the use): 100 μ L/ holes, 37 ℃ of colour developing 15min;
(11) cessation reaction: adding concentration is the sulfuric acid solution color development stopping reaction of 2M, 50 μ L/ holes;
(12) measure, calculate: microplate reader is measured OD
450Value, with blank zeroing, with EDTA and antibody mixed liquor hole as positive control, the OD that positive control hole and well record
450Difference and the OD that records of positive control hole
450Ratio be inhibiting rate, computing formula is as follows:
3. indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury according to claim 2 is characterized in that heavy mercury monoclonal antibody energy specific identification mercury-sequestrant EDTA compound Hg-EDTA, with Zn
2+, Pb
2+, Cd
2+, Al
3+, Ni
2+, Mg
2+, Ca
2+, Co
2+Or Gu
2+Cross reacting rate be lower than 0.13%.
4. according to each described indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury in the claim 1～3, it is characterized in that the CBS with pH=9.6 is cushioned liquid as bag in the reaction system, envelope antigen Hg (II)-ITCBE-BSA extension rate is 1: 5000, the antibody dilution multiple is 1: 80000, best closure is 1% gelatin, EDTA concentration is 5.0mM in the metal mark liquid dilution buffer liquid, the ionic strength of sodium chloride is 0.15mol/L among the antigen-antibody reaction system PBS, optimal pH is 7.4, obtains competition and suppress typical curve under above-mentioned reaction conditions.
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2009
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