CN101526527A - ELISA kit applicable to residue analysis of terrachlor - Google Patents
ELISA kit applicable to residue analysis of terrachlor Download PDFInfo
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- CN101526527A CN101526527A CN200810101552A CN200810101552A CN101526527A CN 101526527 A CN101526527 A CN 101526527A CN 200810101552 A CN200810101552 A CN 200810101552A CN 200810101552 A CN200810101552 A CN 200810101552A CN 101526527 A CN101526527 A CN 101526527A
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- pentachloronitrobenzene
- enzyme
- liquid
- terrachlor
- kit according
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Abstract
The invention provides an ELISA kit applicable to residue analysis of terrachlor, comprising an ELISA plate coated by terrachlor coating antigens, concentrated cleaning solution, a terrachlor standard, terrachlor polyclonal antibodies, ELIAS secondary antibodies, developing solution and stop solution. The terrachlor with known concentration is detected, and a standard curve is drawn, thus the concentration of the terrachlor to be detected can be calculated. The ELISA kit has the advantages of accurately and sensitively detecting terrachlor residues in environment, being capable of detecting a large amount of samples with simple preprocess procedures and least time consumption, having much lower sample-detecting cost than traditional detecting methods.
Description
Technical field
The present invention relates to a kind of Enzyme Linked Immunoadsorbent Assay (ELISA) kit, specifically, relate to a kind of enzyme-linked immuno sorbent assay kit that is applicable to residue analysis of terrachlor.
Background technology
Pentachloronitrobenzene (PCNB) mainly is used as seed dressing and soil sterilants, the multiple diseases that is used to prevent and treat disease in cotton seedling stage, bunt smut and vegetables and fruit tree.Use amount is very big in the cultivation process of Chinese herbal medicine, and especially the medicament as control genseng disease at home and abroad is extensive use of.Pentachloronitrobenzene mainly damages cardiovascular system, central nervous system, liver, kidney and hemopoietic system.The maximum residue limit(MRL) of the mandatory national Specification of China pentachloronitrobenzene in vegetables and raw grain is respectively 0.2 and 0.1mg/kg, and European Union begins to forbid pentachloronitrobenzene January calendar year 2001.
The conventional sense method of pentachloronitrobenzene comprises gas chromatography, high performance liquid chromatography etc., using these physico-chemical analysis technology analyzes the trace pentachloronitrobenzene is residual in the samples such as environment, biology, food, not only the instrumentation degree is had relatively high expectations, and need through complicated separation, extraction, the pre-treatment process such as purify, derive, analysis speed is slow, cost is high, the pre-treatment process need uses a large amount of organic solvents, has caused new environmental pollution again.Along with sample to be checked, particularly require increasing sharply of field quick detection sample size, traditional pesticide residue analysis means are difficult to adapt to requirement, therefore, press for development and application high-level efficiency residues of pesticides express-analysis technology.
Summary of the invention
(1) technical matters that will solve
At above-mentioned deficiency, the invention provides a kind of have high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, the simple enzyme-linked immuno sorbent assay kit of method of operating, be used for the residual batch of environment pentachloronitrobenzene, fast detecting.
(2) technical scheme
For achieving the above object, the invention provides a kind of residue analysis of terrachlor enzyme-linked immuno sorbent assay kit, this kit comprises by the ELISA Plate of pentachloronitrobenzene envelope antigen, concentrated cleaning solution, pentachloronitrobenzene standard items, pentachloronitrobenzene specific antibody, enzyme labeling thing, substrate colour developing liquid, reaction terminating liquid.
Wherein, used immunogene is the coupled complex of pentachloronitrobenzene haptens and carrier protein in the preparation process of pentachloronitrobenzene antibody, described carrier protein can be common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin, is preferably bovine serum albumin(BSA); Described polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, is preferably rabbit source polyclonal antibody.
Wherein, bag is by in the preparation process of ELISA Plate of pentachloronitrobenzene envelope antigen, used envelope antigen is the coupled complex of pentachloronitrobenzene haptens and ovalbumin, used coating buffer can be 0.05M pH 9.6 sodium carbonate buffers, and used confining liquid is the above-mentioned coating buffer that contains 1% gelatin.
Wherein, the enzyme labeling thing is an ELIAS secondary antibody, and marker enzyme can be horseradish peroxidase or alkaline phosphatase.
Wherein, the prescription of concentrated cleaning solution is to add sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL in every 20mL distilled water.The concentration of concentrated cleaning solution is 50 times when normally using.
When marker enzyme was horseradish peroxidase, substrate colour developing liquid was made up of A liquid and B liquid, and colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L.Specifically, the A formula of liquid can be and adds urea peroxide 1g, 10.3g citric acid, 35.8g Na in every 1000mL water
2HPO
412H
2O, Tween-20 100 μ L, pH5; The B formula of liquid can be and adds tetramethyl benzidine (TMB) 700mg (40mL DMSO dissolving), 10.3g citric acid, pH2.4-2.6 in every 1000mL distilled water.When marker enzyme is a bacterium when extracting alkaline phosphatase, substrate colour developing liquid is for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
The analysis principle of kit of the present invention is:
When the pre-envelope antigen of ELISA Plate, after adding pentachloronitrobenzene sample to be measured and pentachloronitrobenzene polyclonal antibody, solid-phase coating antigen and pentachloronitrobenzene to be measured are vied each other and antibody response, because the solid phase antigen in each hole and the antibody content of adding are all consistent, so when pentachloronitrobenzene concentration to be measured is high, the antibody that then is bonded on the solid phase antigen is few, the ELIAS secondary antibody that adds is few with the antibodies amount that is fixed, add substrate colour developing liquid with cleansing solution washing back, chromogenic reaction is shallow, the OD value that detects with microplate reader is low, shows the inhibiting rate height; Otherwise when pentachloronitrobenzene concentration to be measured was low, the OD value of then being surveyed was high, and inhibiting rate is low.According to detecting the typical curve of being done, can extrapolate the concentration of pentachloronitrobenzene to be measured with known pentachloronitrobenzene concentration.
(3) beneficial effect
Advantage of the present invention be can be accurately the pentachloronitrobenzene in the testing environment is residual delicately, the pre-treatment process of sample is simple, and is consuming time few, can detect a large amount of samples simultaneously, the sample detection cost is far below traditional instrument detecting method.The present invention has important practical significance to the residual on-site supervision technology of pentachloronitrobenzene that solves batch samples.
Embodiment
Following examples are used for further specifying of the present invention, but are not used for limiting invention which is intended to be protected.
Operation of embodiment 1 kit and result calculate
Testing sample is after pre-treatment, and is standby with the PBST constant volume.Take vacuum packaging bag apart and take out ELISA Plate, at room temperature balance 5 minutes is standby.Preparation 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, the pentachloronitrobenzene titer of 2000ng/mL, add 50 μ L standard specimens or the sample handled well in each hole, standard specimen and sample are done 4 repetitions, add the antibody of 50 μ L dilution, hatch 30 minutes for 37 ℃; Pour out the liquid in the hole, the PBST good with dilution washes 5 times, ELISA Plate is upside down in pat dry on the thieving paper; Add by the good enzyme mark goat-anti rabbit two anti-100 μ L of dilution in 1: 1000, hatched 30 minutes for 37 ℃; Pour out the liquid in the hole, wash plate 5 times, pat dry with PBST; Get A liquid and B liquid equal-volume mixing, every hole adds 100 μ L, and 37 ℃ of colour developings 30 minutes, every hole added the stop buffer cessation reaction of 50 μ l, and measuring each hole on the microplate reader is the OD value at 450nm place at wavelength.
The OD value that will contain 0ng/mL standard items hole deducts the OD value that contains Cmax standard items hole and is decided to be B
0, the OD value after all the other apertures are proofreaied and correct with quadrat method is decided to be B; With B/B
0Value is ordinate, and the log value of respective standard product concentration is a horizontal ordinate, draws the pentachloronitrobenzene standard and suppresses curve.Can obtain the concentration of counter sample according to the regression equation of curve, also can obtain pentachloronitrobenzene suppress in concentration IC
50(B/B
0=50%) and minimum detectable level IC
20(B/B
0=80%).Wherein, concentration IC in the inhibition
50=37ng/ml, lowest detectable limit IC
20=7.2ng/ml.
Embodiment 2 is haptenic synthetic
Take by weighing the 2g pentachloro-phenol (pentachlorophenol, PCP) and 1gK
2CO
3Be dissolved in the 30mL acetone, join in the reactor, under protection of nitrogen gas, add the 1.14g bromo-propionic acid, the back flow reaction of heating 2 hours adds the 0.5g bromo-propionic acid again, keeps the constant reaction of above condition spend the night (gas that reaction generates feeds the NaOH absorption bottle and absorbs).Remove organic solvent under reduced pressure, surplus materials separates with concentrated hydrochloric acid and methylene chloride, discards inorganic phase, the organic phase anhydrous Na
2SO
4Drying, remove organic solvent under reduced pressure after, purify with silica gel column chromatography and promptly to obtain the thick product of required haptens.Yield 50%.
The preparation of embodiment 3 pentachloronitrobenzene polyclonal antibodies
With active ester method with haptens and bovine serum albumin(BSA) coupling, claim the 2mg conjugate to be dissolved in the 1mL physiological saline, mix with the 1mL complete Freund's adjuvant, New Zealand's large ear rabbit thigh is injected in fully emulsified back, later on every two all booster immunizations once, use incomplete Freund's adjuvant instead and mix with immunogene, immune position is that nape portion is subcutaneous, from immunity for the third time, each immunity one week of back is detected serum titer from the rabbit ear vein blood sampling.Immunity is 5 times altogether, and whole blood was adopted from rabbit arteria carotis in one week in last immunity back, slightly carried rabbit anti-serum with 35% saturated ammonium sulfate salting out method, was further purified with the DE-52 anion exchange chromatography at last, obtained purer pentachloronitrobenzene polyclonal antibody.
DE-52 anion exchange antibody purification:
(1) DE-52 pre-service: need 5g weight in wet base cellulose to calculate with the thick IgG of 1mg, claim an amount of DE-52 to add 0.5mol/LNaOH and soak 30min, distilled water is washed till neutrality repeatedly; Then soak 30min with 0.5mol/LHCl, distilled water is washed till neutrality repeatedly; Soak 30min with 0.5mol/LNaOH again, distilled water is washed till neutrality repeatedly, uses the abundant equilibrate overnight of PBS of 0.01mol/L, pH7.4 at last.
(2) dress post: take out bubble in the DE-52 solution with vacuum pump before the dress post, can not produce bubble in the dress post process, the last sample front pillar bed PBS balance of 0.01mol/L, pH7.4.
(3) go up sample: the PBS with 4～6mL dilutes an amount of thick IgG, slowly is added in post bed upper surface along the post arm then, treats to begin wash-out after sample enters the post bed.
(4) wash-out: the PBS with 0.01mol/L, pH7.4 makes eluent, and flow velocity 0.5mL/min collects first and second absorption peaks.
(5) collection liquid cooling freeze-drying is dry, 4 ℃ of preservations.
The preparation of embodiment 4 ELISA Plate
Be cushioned liquid (0.05M pH 9.6 sodium carbonate buffers) with bag the pentachloronitrobenzene antigen diluent is become 1 μ g/mL, every hole adds 100 μ L, 37 ℃ of incubation 2h and 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution washing 3 times, each 30 seconds, pat dry, in every hole, add 150-200 μ L confining liquid then, 37 ℃ of incubation 2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
In this example of establishment of embodiment 5 residue analysis of terrachlor enzyme-linked immuno sorbent assay kits, kit comprises as the lower part:
(1) bag is by the ELISA Plate of pentachloronitrobenzene antigen
(2) sponge bracket
(3) 1mg/mL pentachloronitrobenzene standard items
(4) pentachloronitrobenzene polyclonal antibody
(5) horseradish peroxidase mark goat anti-rabbit antibody
(6) the thickening and washing formula of liquid is: sodium chloride 8g, potassium dihydrogen phosphate 0.2g, sodium hydrogen phosphate 3g, potassium chloride 5g, Tween-20 2mL, distilled water 20mL.
(7) colour developing liquid A formula of liquid: urea peroxide 1g, 10.3g citric acid, 35.8g Na
2HPO
412H
2O, Tween-20 100 μ L, distilled water 1000mL, pH5.
(8) colour developing liquid B formula of liquid: tetramethyl benzidine (TMB) 700mg (40mL DMSO dissolving), 10.3g citric acid, distilled water 1000mL, pH2.5.
The experiment of embodiment 6 kit specificitys
Use the kit among the embodiment 5, experimentize.Select analogue pentachloro-phenol, the hexachloro-benzene, 2 of pentachloronitrobenzene, 4-D, pentachloro-phenol ethyl ester, PCB, haptens, esterification haptens be as determinand, records concentration (IC in the inhibition of various materials
50), use the cross reactivity of following formula calculating antibody again to these materials; Cross reacting rate is littler, and then antibody is stronger to the specificity of pentachloronitrobenzene, on the contrary the poor specificity of antibody then.
Cross reaction (CR%)=IC
50(pentachloronitrobenzene)/IC
50(for the examination thing) * 100%.
Measuring the results are shown in Table 1, adopt indirect elisa method, pair there is certain cross reaction in polyclonal antibody with the pentachloronitrobenzene analogue, and polyclonal antibody is to the cross reaction of hexachloro-benzene more greatly 12%, and less to the cross reaction of synthetic haptenic raw material pentachloro-phenol (PCP).The cross reaction of the PCP of haptens, esterification haptens, esterification is respectively 230%, 15% and 8%, but these materials do not exist at occurring in nature, and antibody can not be subjected to the interference of above-mentioned substance.Close or similar substance does not then all have tangible cross reaction to other structure.The specificity that this kit is described is good, can guarantee the reliability to pentachloronitrobenzene determined result of residue in the sample.
Table 1 cross reaction
Table1?Cross?Reactivities?of?Indirect?ELISA?Kit
Embodiment 7 recovery tests
Use the kit among the embodiment 5, experimentize.After well water, river, soil add pentachloronitrobenzene,, the results are shown in Table 2 with its content of kit measurement.
Add the ELISA measurement result of pentachloronitrobenzene in table 2 well water, river, the soil
Claims (8)
1. an enzyme-linked immuno sorbent assay kit that is applicable to residue analysis of terrachlor comprises that bag is by the ELISA Plate of pentachloronitrobenzene antigen, concentrated cleaning solution, pentachloronitrobenzene standard items, pentachloronitrobenzene specific antibody, enzyme labeling thing, substrate colour developing liquid and reaction terminating liquid.
2. enzyme-linked immuno sorbent assay kit according to claim 1, wherein, pentachloronitrobenzene antibody used immunogene in preparation process is the coupled complex of pentachloronitrobenzene haptens and carrier protein BSA; Described pentachloronitrobenzene antigen is the coupled complex of pentachloronitrobenzene and carrier protein OVA.
3. enzyme-linked immunologic detecting kit according to claim 1 and 2, wherein, described enzyme labeling thing is an ELIAS secondary antibody.
4. enzyme-linked immunologic detecting kit according to claim 3, wherein, described ELIAS secondary antibody is the horseradish peroxidase-labeled goat anti-rabbit antibody.
5. enzyme-linked immuno sorbent assay kit according to claim 1 and 2, wherein, the prescription of concentrated cleaning solution is to add sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL in every 20mL distilled water.
6. enzyme-linked immuno sorbent assay kit according to claim 1 and 2, it is characterized in that, when marker enzyme is horseradish peroxidase, colour developing liquid comprises A liquid and B liquid, and described A liquid is that hydrogen peroxide or urea peroxide, described B liquid are that o-phenylenediamine or tetramethyl benzidine, stop buffer are sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme is a bacterium when extracting alkaline phosphatase, substrate colour developing liquid is for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
7. enzyme-linked immuno sorbent assay kit according to claim 6, wherein, described A formula of liquid is to add urea peroxide 1g, 10.3g citric acid, 35.8g Na in every 1000mL water
2HPO
412H
2O, Tween-20 100 μ L, pH5; Described B formula of liquid is to add tetramethyl benzidine 700mg, 10.3g citric acid, pH2.4-2.8 in every 1000mL distilled water.
8. the application of each described detection kit of claim 1～7 in the detection pentachloronitrobenzene is residual.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810101552A CN101526527A (en) | 2008-03-07 | 2008-03-07 | ELISA kit applicable to residue analysis of terrachlor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810101552A CN101526527A (en) | 2008-03-07 | 2008-03-07 | ELISA kit applicable to residue analysis of terrachlor |
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| Publication Number | Publication Date |
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| CN101526527A true CN101526527A (en) | 2009-09-09 |
Family
ID=41094503
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|---|---|---|---|
| CN200810101552A Pending CN101526527A (en) | 2008-03-07 | 2008-03-07 | ELISA kit applicable to residue analysis of terrachlor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104678105A (en) * | 2015-01-16 | 2015-06-03 | 镇江市第一人民医院 | Enzyme linked immunosorbent assay kit for detecting Cripto-1 and preparation method thereof |
| CN105301251A (en) * | 2014-07-25 | 2016-02-03 | 江苏维赛科技生物发展有限公司 | Preparation and using method of quintozene colloidal gold test strip |
| CN111735952A (en) * | 2020-06-04 | 2020-10-02 | 北京勤邦生物技术有限公司 | Test strip and method for detecting hexachlorobenzene |
| CN113281501A (en) * | 2021-05-12 | 2021-08-20 | 北京勤邦生物技术有限公司 | Application of pentachloronitrobenzene artificial antigen in enzyme linked immunosorbent assay kit |
-
2008
- 2008-03-07 CN CN200810101552A patent/CN101526527A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301251A (en) * | 2014-07-25 | 2016-02-03 | 江苏维赛科技生物发展有限公司 | Preparation and using method of quintozene colloidal gold test strip |
| CN104678105A (en) * | 2015-01-16 | 2015-06-03 | 镇江市第一人民医院 | Enzyme linked immunosorbent assay kit for detecting Cripto-1 and preparation method thereof |
| CN111735952A (en) * | 2020-06-04 | 2020-10-02 | 北京勤邦生物技术有限公司 | Test strip and method for detecting hexachlorobenzene |
| CN111735952B (en) * | 2020-06-04 | 2023-07-11 | 北京勤邦科技股份有限公司 | Test strip and method for detecting hexachlorobenzene |
| CN113281501A (en) * | 2021-05-12 | 2021-08-20 | 北京勤邦生物技术有限公司 | Application of pentachloronitrobenzene artificial antigen in enzyme linked immunosorbent assay kit |
| CN113281501B (en) * | 2021-05-12 | 2023-07-07 | 北京勤邦科技股份有限公司 | Application of pentachloronitrobenzene artificial antigen in ELISA kit |
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Inventor after: Xu Ting Inventor after: Li Ji Inventor after: Shao Xiaolong Inventor before: Li Ji Inventor before: Xu Ting Inventor before: Shao Xiaolong |
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Application publication date: 20090909 |