CN103487584A - Enzyme-linked immunosorbent assay (ELISA) detection method for perfluorooctane sulfonate residual in environment - Google Patents
Enzyme-linked immunosorbent assay (ELISA) detection method for perfluorooctane sulfonate residual in environment Download PDFInfo
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Abstract
The invention relates to an enzyme-linked immunosorbent assay (ELISA) detection method, which can be applied to enzyme-linked immunosorbent assay (ELISA) detection of perfluorooctane sulfonated (PFOs) residual in the environment. The method comprises following steps: step one, synthesizing and identifying PFOs artificial antigen; step two, preparing and purifying PFOs antibody; step three, establishing PFOs indirectly competing ELISA quantitative detection method; step four, substituting PFOs with 9 PFCs with similar structures as that of PFOs, and then respectively measuring the cross-reactivity of the 9 PFCs with an antibody. The technology provides a method of rapidly detecting PFOs residual in the environment, better improves the detection sensitivity, guarantees the precision of the detection results, satisfy the requirements on PFOs residual detection at present, and is convenient for carrying out PFOs residual detection in the environment.
Description
Technical field
The present invention relates to a kind of enzyme-linked immune detection method, can be applicable to the enzyme linked immunosorbent detection residual to Perfluorooctane sulfonates in environment.
Background technology
Perfluorochemical (Perfluorinated compounds, PFCs) good thermal stability, chemical stability, high surface and the hydrophobic oleophobic performance with it, be widely used in many commercial production such as polymeric additive, lubricant and daily necessities.Perfluorooctane sulfonates (Perfluorooctane Sulphonate, PFOs) and perfluoro caprylic acid (Perfluorooctanoic Acid, PFOA) are representational PFCs.In May, 2009 United Nations Environment Programme (UNEP) and " about the Convention of Stockholm of persistence organic pollutant (POPs) " the 4th conference of contracting party just reduces and finally bans use of the toxic chemical substance of 9 kinds of serious harm human healths and physical environment to reach common understanding, and these 9 kinds of new POPs just comprise PFOs and derivant perfluoro octane sulfonate and full-fluorine octyl sulfuryl fluoride.Because the C-F bond energy of PFCs is very big, be difficult to degraded under the home condition, can not effectively be removed by traditional sewage water treatment method, so the global environmental pollution that PFCs causes has become the fact.Between Alexander G.P. estimation 1970-2002, the output of global perfluoro capryl sulfonyl compound is ten thousand tons of 9.6-12.25, and wherein in sea surface, amount is estimated as the 235-1770 ton.
Ripe not enough for the analytical approach of PFOs, the analytical approach of complex environment, biology and food samples needs further perfect, and Criterion method gradually.In the research of PFCs analytical approach, vital link is the pre-treatment of sample.Because PFCs has the duality of hydrophobic oleophobic, belong to the trace amount environment pollutant, and environmental matrices is as soil, biological sample complicated component all often, so sample pre-treatments difficulty has comparatively become one of key factor of restriction environmental monitoring and this class physical environment behavior of further investigation.
At environmental science, immunoassay technology research has obtained certain achievement, Weilin L.Shelver has reported the Enzyme-linked Immunosorbent Assay method (ELISA) detected for environment PBDE (PBDEs) recently, and detectability and GC-NCI-MS are suitable, can reach 0.1ug/L.Data shows that ELISA can be used for the PBDEs detection of environment, biological sample, and the recovery and instrumental method are suitable.The residue analysis method of PFOs is the LC-MS method at present, also there is no the ELISA method report of relevant PFOs.Although these methods can reach the requirement of retention analysis, the sample pretreatment process complexity, workload is large, the problems such as instrument costliness, and the ELISA method is highly sensitive, high specificity, sample pre-treatments is simple, easy to operate, be convenient to carry out on-site supervision, can complement one another with conventional method of analysis.
Summary of the invention
The objective of the invention is to develop a kind of accuracy and precision high, be applicable to the residual enzyme-linked immune detection method of Perfluorooctane sulfonates in environment.
Specific implementation step of the present invention is as follows:
Synthetic and the evaluation of 1PFOs artificial antigen
Take 150mg (0.3mmol) PFOS and 60mg N-hydroxy-succinamide (0.4mmol) is put in reaction bulb, add the 2ml dry DMF and dissolve.Separately get 85mg N, N '-dicyclohexylcarbodiimide (0.4mmol) is dissolved in the 2ml dry DMF, and this solution is added to reaction bulb, stirring at room reaction 5 hours.Reactant liquor is put under 4 ℃ of conditions airtight standing more than 2 hours.Centrifugal, get supernatant active ester liquid.Take carrier protein bovine serum albumin(BSA) (BSA), each 60mg of ovalbumin (OVA), be dissolved in respectively the phosphate buffer (PB) of 4ml 0.05mol/L pH8.0.Under stirring, the gradation of above-mentioned active ester liquid equivalent is added in protein solution, approximately within 30 minutes, adds.The slow stirring reaction of room temperature one hour, 4 ℃ of slow stirring reactions spend the night.Put reactant liquor in bag filter next day, and in the PB of 0.01mol/L pH6.8,4 ℃ are stirred dialysis, within every 4 hours, change dislysate one time, until do not measure haptens in dislysate.After the accurate measurement dialysis, the volume of conjugate solution, calculate protein concentration.Make blank with last extracellular fluid dialysis, respectively conjugate solution, haptens solution, carrier protein solution are carried out to UV scanning, according to scanning spectra, determine whether haptens with carrier protein, coupling has occurred.Calculate respectively molar absorptivity ε 290 according to OD290 and concentration separately, and the ratio of the estimation haptens molecular number of being combined with carrier protein (in conjunction with than).Conjugate solution is sub-packed in cryopreservation tube ,-20 ℃ of preservations.
The preparation and purification of 2 antibody
Select to make immunogene in conjunction with the conjugate PFOs-BSA than 10～20/1, PFOs-OVA makes coating antigen.First immunisation adopts the immunogene by Freund's complete adjuvant emulsification, in the immunity of rabbit back intracutaneous multi-point injection.Interval surrounding, the later immunogene of using by incomplete Freunds adjuvant emulsification every two weeks after first immunisation, in rabbit back and the subcutaneous multi-point injection booster immunization of neck, immunizing dose is that per kilogram rabbit body weight is respectively 200,400,600,800ug (in carrier protein).Since the 4th time, latter one week of each immunity, rabbit ear edge vein is adopted a small amount of blood, separation of serum.Agar double immunodiffusion method measure antiserum titre reach 1: 16 above, indirect elisa method measure mid point tire the OD value ≈ 1.0 of the enzyme-catalytic chromogenic reaction product (time sero-fast extension rate) reach at 2 * 104 o'clock, rabbit arteria carotis is adopted whole blood, separation of serum.
If finally obtain the pooled serum of 1 volume, add the physiological saline of equivalent, then the stirring of dropping limit, limit, add altogether 2 volume saturated ammonium sulfates, make it to reach 50% saturation degree, more than with strong aqua, the pH value being adjusted to 7.0,40 ℃ of heating 30min, balance is to room temperature.Then with the centrifugal 30min of 3000 turn/min.Abandon supernatant, add 2 volume physiological saline, dissolution precipitation thing (being mainly Immunoglobulin IgG), dropwise add 1 volume saturated ammonium sulfate, makes it to reach 30% saturation degree, and the pH value is adjusted to 7.0, centrifugal treating after equilibrium at room temperature, and this step repeats 2-3 time.The a small amount of normal saline dilution of the centrifugal sediment finally obtained.
Sediment by normal saline dilution after, become milky white solution.For removing the ammonium sulfate in the immunoglobulin (Ig) crude extract, the bag filter of it should being packed into, first use distill water dialysis 12hr, during change liquid 2-3 time, then, with the phosphate-buffered salt of 0.01M pH7.2 dialysis 12hr, change liquid 2-3 time.The volume of dislysate is that 40 times of serum volume are advisable, and whole dialysis procedure need to continuous gentle agitation complete under 4 ℃ of conditions.
The foundation of the indirect competitive ELISA quantitative detecting method of 3PFOS
(1) take the PFOs-OVA of a certain amount of concentration is envelope antigen, coated 96 hole ELISA Plate, and every hole 100 μ l, 37 ℃ of incubation 2hr, dry, and with cleansing solution washing 3 times, adds standing interval 3min after cleansing solution at every turn, gets rid of to anhydrate minute, on thieving paper, pats dry.
(2) with 1% OVA sealase target, every hole 200 μ l, 37 ℃ of incubation 1hr, wash 3 times, pats dry.
(3) by certain concentration gradient dilution PFCs standard specimen, every hole adds 50 μ l, then adds the 50 μ l anti-PFOs-BSA antibody of homemade rabbit (primary antibodie) in every hole, and 37 ℃ of incubation 1h, wash 5 times, pats dry.
(4) every hole adds the 100 μ l goat-anti rabbit-HRP of 1: 1000 (two is anti-), and 37 ℃ of incubation 1hr, wash 5 times, pats dry.
(5) OPD-H that adds 100 μ l/ holes now to join
2o
2substrate solution, 37 ℃ of standing 15～20min chromogenic reactions of lucifuge, then every hole adds the concentrated sulphuric acid cessation reaction of 50 μ l 2M, by microplate reader, detects OD492nm.
The present invention is screened envelope antigen and antibody working concentration, selects envelope antigen concentration 0.1ug/ml, antibody dilution 1: 8000.
The present invention is diluted to a series of concentration gradients by 9 kinds of chemical constitution PFCs or catabolite standard specimens (purity 98%) similar to PFOs, replace the PFOs standard specimen, measure respectively the cross reacting rate of itself and antibody: antibody is except the cross reacting rate higher (16.2% with perfluoro decane sulfonic acid (PFDS) and perflexane sulphur (PFHxS), 14.22%) outside, all be less than 10% with the cross reacting rate of other compound, so antibody prepared by the present invention there is specificity preferably.
the accompanying drawing explanation
Accompanying drawing 1 means the standard ELISA curve of different antibodies.
Embodiment
Below in conjunction with embodiments of the invention, elaborate: the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The mensuration of embodiment 1PFOs standard specimen
The PFOs standard sample dilution is become to 1,10,50,100,500,1000,5000ug/l and 7 concentration gradients of blank, and it is blank that serum antibody is established carbonate buffer solution.The best antigen, the antibody working concentration that use the present invention to filter out carry out indirect competitive enzyme-linked immunosorbent mensuration, prepare the antibody typical curve that 4 rabbits produce.If the OD value of antibody blank is ODmin, the OD value of PFCs blank is ODmax, ordinate inhibiting rate=[(ODmax-ODmin)-(ODmax-OD is to be measured)]/ODmax-ODmin.Horizontal ordinate is got the logarithm of PFCs concentration, according to corresponding OD492 value, draws out typical curve, sees accompanying drawing 1.
The standard specimen measurement result shows: 4 antibody typical curve indifferences that rabbit produces, between 10-1000ug/l, curve is the good linear relation, and this section rate of curve is larger, so linear detection range is 10-1000ug/l.When PFOs concentration reaches 1ug/l, the OD value of its OD value and PFCs blank presents notable difference, therefore PFOs detects, is limited to 10ug/l.
Embodiment 2 is used enzyme-linked immunoassay method to detect the recovery of standard addition of 4 kinds of different quality water samples
The water sample of 4 kinds of different qualities is added respectively to 10,50, the PFOs standard specimen that 3 kinds of concentration of 100ug/ml are different, every kind of concentration is established 3 repetitions, using distilled water as blank, for the enzyme-linked immune detection method analysis, check in the content of PFOs according to the OD value recorded on typical curve, calculate the recovery, the results are shown in subordinate list 1.
In the linear detection range 10-1000ug/l of method, the average recovery rate of PFOs is 101.3%, and average coefficient of variation is 5.93%.
The interpolation recovery of different samples under subordinate list 1 screening conditions of the present invention
Claims (6)
1. the residual enzyme-linked immune detection method of Perfluorooctane sulfonates in an environment, it is characterized in that, with manually synthetic Perfluorooctane sulfonates immunogen immune new zealand white rabbit, prepare polyclonal antibody, with sad-ammonium sulfate precipitation method antibody purification, selecting envelope antigen concentration is 0.1ug/ml, and antibody dilution is 1: 8000.
2. polyclonal antibody according to claim 1, is characterized in that, selects to make immunogene in conjunction with the conjugate PFOS-BSA than 10～20/1, and PFOS-OVA makes coating antigen.Use immunogene to carry out immunity to animal, interval surrounding, the later immunogene of using by incomplete Freunds adjuvant emulsification every two weeks after first immunisation.In the 4th time, latter one week of each immunity, adopt a small amount of blood, separation of serum.
3. immune animal according to claim 2 is New Zealand white rabbit, rabbit 5-6 in age month, and body weight 2.5～3kg.
4. polyclonal antibody according to claim 1, it is characterized in that, this antibody is except the cross reacting rate higher (16.2% with perfluoro decane sulfonic acid (PFDs) and perflexane sulphur (PFHxs), 14.22%) outside, all be less than 10% with the cross reacting rate of other compound, show that this antibody has specificity preferably.
According to claim 1 sad-the ammonium sulfate precipitation method antibody purification, it is characterized in that, add saturated ammonium sulfate, with strong aqua, the pH value is adjusted to 7.0, centrifugal treating after equilibrium at room temperature.Repeat this step 2-3 time.
6. the residual enzyme-linked immune detection method of Perfluorooctane sulfonates in a kind of environment according to claim 1, it is characterized in that, the linear detection range of method is 10-1000ug/l, the detection of PFOs is limited to 10ug/l, in this scope, average recovery rate is 101.3%, and average coefficient of variation is 5.93%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118015A (en) * | 2020-01-19 | 2020-05-08 | 华侨大学 | Preparation and application of potassium perfluorooctane sulfonate resistant aptamer |
| CN111542497A (en) * | 2017-08-28 | 2020-08-14 | 纽卡斯尔大学 | Adsorbent and process for producing the same |
-
2012
- 2012-06-11 CN CN201210190645.7A patent/CN103487584A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111542497A (en) * | 2017-08-28 | 2020-08-14 | 纽卡斯尔大学 | Adsorbent and process for producing the same |
| CN111118015A (en) * | 2020-01-19 | 2020-05-08 | 华侨大学 | Preparation and application of potassium perfluorooctane sulfonate resistant aptamer |
| CN111118015B (en) * | 2020-01-19 | 2022-07-29 | 华侨大学 | Preparation and application of potassium perfluorooctane sulfonate-resistant aptamer |
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