CN105738611A - Benzo(a)pyrene enzyme-linked immunosorbent assay (ELISA) kit and detection method thereof - Google Patents
Benzo(a)pyrene enzyme-linked immunosorbent assay (ELISA) kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses an ELISA kit for detecting residual benzo(a)pyrene. The ELISA kit includes a 96-well ELISA plate coated by a benzo(a)pyrene antigen, a benzo(a)pyrene specific antibody, an ELISA secondary antibody, a benzo(a)pyrene reference substance solution, a sample diluent, an antibody diluent, a color developing agent and a stopping solution. The invention also discloses a method of detecting the benzo(a)pyrene in water and soil samples with the kit, wherein the method includes the steps of pre-treating the sample, detecting the sample with the kit and processing and analyzing a result. The kit for detecting the residual benzo(a)pyrene employs the indirect competitive ELISA technology, has high sensitivity and good stability, is low in cost, and is very suitable for detection on large quantity of samples and has important promoting application value.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, it is specifically related to a kind of for detecting the enzyme linked immunological kit of benzo (a) pyrene residual in soil and water, it is particularly well-suited to Drinking Water, subsoil water, water of river and lake, the detection of benzo (a) pyrene residual in the water sample such as industrial wastewater and contaminated soil.
Technical background
Benzo (a) pyrene (Benzoapyrene, BaP) is the polycyclic aromatic hydrocarbon containing 5 phenyl ring, is the maximum a kind of carcinogen of polycyclic aromatic hydrocarbon toxic, produces under the imperfect combustion state between 300 to 600 DEG C.Therefore, benzo (a) pyrene becomes the encountered great environmental in countries in the world and one of public health problem.Benzo (a) pyrene is classified as priority pollutants by American National Environmental Protection Agency (USEPA), and it is carried out strict supervision and control.The concentration limitation of benzo (a) pyrene in strict regulations surface water in China's water environment quality standard (GB3838-2002).
In environmental sample benzo (a) pyrene monitoring and analyze mainly include following step: sample collecting, benzo (a) pyrene extracts, purify and concentration, finally adopt the instrument analytical method such as gas chromatography or high performance liquid chromatography to detect.Whole process steps is loaded down with trivial details, consuming time longer, and instrument and equipment is expensive, is not suitable for the analysis in the short time of a large amount of samples and monitoring.Except the instrument analytical method that both the above is main, the method being used for measuring benzo (a) pyrene also has fluorescence method and enzyme linked immunosorbent assay analysis method.Compared with other method, enzyme linked immunosorbent assay analysis method is applied to the detection analysis of environmental contaminants early has precedent abroad, and obtains the accreditation of external environmental protection department.Enzyme linked immunosorbent assay analysis method (Enzyme-LinkedImmunoSorbentAssay, ELISA) is that the immunoreation of Ag-Ab and the efficient catalytic reaction of enzyme are organically combined a kind of integrated technology formed.There is specificity simultaneously good, highly sensitive, simple and efficient to handle, and the melancholy points such as batch samples can be processed simultaneously.Therefore, benzo (a) pyrene is carried out detection analysis and increasingly receives the concern of research worker by research application the method.
Summary of the invention
It is an object of the invention to the method overcoming existing detection benzo (a) pyrene the dependency of equipment is high, and the shortcoming that the quickly detection to batch samples can not be realized, there is provided enzyme-linked immunologic detecting kit and the detection method thereof of benzo (a) pyrene in soil and water in one detection environment, the restriction of the not examined equipment of the present invention and be capable of large batch of benzo (a) pyrene residual sample is carried out rapid screening.
Another object of the present invention is to provide a kind of quick, the easy qualitative or quantitative method of benzo (a) pyrene in detection sample.
To achieve these goals, the present invention adopts following measuring principle: first by benzo (a) pyrene Antigen adsorption in ELISA Plate hole, it is subsequently adding testing sample and benzo (a) the pyrene antibody of artificial preparation, second antibody (the ELIAS secondary antibody for first antibody with enzyme labelling, if primary antibodie is rabbit antibody, then ELIAS secondary antibody is enzyme mark goat anti-rabbit igg antibody；If primary antibodie is murine antibody, then ELIAS secondary antibody is enzyme mark sheep anti-mouse igg antibody), antigen in ELISA Plate hole is vied each other and benzo (a) the pyrene antibodies added with the determinand in sample, after washing, in ELISA Plate hole, leave the conjugate that antigen-benzo (a) pyrene enzyme of antibody mark two is anti-.Washing away unconjugated benzo (a) pyrene antibody and ELIAS secondary antibody, add substrate, substrate for enzymatic activity is reacted and is developed the color.Antigen fixing in ELISA Plate and the amount of the primary antibodie of addition, ELIAS secondary antibody are certain, if testing concentration is higher in sample, then required amount of antibody in combination is just more, and the amount of antibody that antigen is combined on carrier is just relatively fewer, color reaction weakens, and absorbance reduces；Otherwise, then color reaction strengthens, and absorbance increases.Thus, the standard curve of benzo (a) pyrene according to concentration known and the absorbance of testing sample, map further according to the semilog relation between absorbance and benzo (a) pyrene concentration and obtain standard curve, and extrapolating the concentration of benzo (a) pyrene in testing sample.
Above-mentioned purpose of the present invention can be achieved through the following technical solutions:
Enzyme linked immunological kit provided by the invention includes following components:
It is coated the ELISA Plate of benzo (a) pyrene antigen
Benzo (a) pyrene standard solution
Benzo (a) pyrene antibody working solution
ELIAS secondary antibody
Antibody diluent
Sample diluting liquid
Nitrite ion
Stop buffer
Described benzo (a) pyrene antigen is obtained by activity fat method coupling by benzo (a) pyrene hapten and carrier protein, and described carrier protein is bovine serum albumin, oralbumin；Used is coated the carbonate buffer solution that liquid is pH9.6,0.05M, and the concentration that is coated of benzo (a) pyrene antigen is 0.5～1 μ g/ml, and confining liquid is preferably the phosphate buffer (PBS solution) containing 2%BSA.Described PBS solution concentration is 0.01mol/L, pH value 7.4, and wherein Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
Described ELISA Plate is 96 hole polystyrene ELISA Plate, is coated with benzo (a) the pyrene antigen being combined with anti-benzo (a) pyrene antibody specificity, and the site of benzo (a) pyrene antigen is not adsorbed on closed porosity plate surface.
Described specific antibody is the one in benzo (a) pyrene polyclonal antibody or monoclonal antibody, prepares for this laboratory early stage immunization method routinely, collects ascites and adopts affinity column to be purified.
Described ELIAS secondary antibody is sheep anti mouse or the goat anti-rabbit antibody of horseradish peroxidase-labeled.
Described benzo (a) pyrene standard solution concentration respectively 0ppb, 4ppb, 20ppb, 100ppb, 500ppb.
Described antibody diluent is that wherein phosphate buffer Contents of Main Components is KH containing 1% bovine serum albumin and 0.01% polysorbas20, the phosphate buffer of pH7.42PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
Described sample diluting liquid is that wherein phosphate buffer Contents of Main Components is KH containing 30% methanol, 1%～5% bovine serum albumin and 0.05% polysorbas20, the phosphate buffer of pH7.42PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
Described substrate nitrite ion is the one-component nitrite ion containing TMB (TMB).Described stop buffer is 1～2mol/L sulfuric acid solution.
The preparation method of the ELISA Plate being coated with benzo (a) pyrene antigen of the present invention:
With being coated liquid, benzo (a) pyrene antigen is diluted on demand, in ELISA Plate hole, add antigenic dilution, put into 4 DEG C of environment night incubation, clean and be coated liquid, washing, then more every hole adds confining liquid, room temperature 25 DEG C is hatched 1 hour, and dried sealing by aluminium foil bag vacuum preserves.
Described benzo (a) pyrene antibody specific preparation method is presented below:
(1) synthesis of benzo (a) pyrene antigen: by benzo (a) pyrene derivatives as hapten respectively with the carrier protein such as bovine serum albumin (BSA) and oralbumin (OVA) by activity fat method coupling, obtain immunogen BAP-BSA and coating antigen BAP-OVA.
(2) animal immune: adopt Balb/C mice as immune animal, with immunogen BAP-BSA, mice is carried out immunity, it is possible to obtain the mouse spleen containing benzo (a) pyrene specific antibody in blood.
(3) cell fusion and clone: take splenocyte and the myeloma cell fusion of immunity Balb/C mice, adopts Indirect cELISA to measure cell conditioned medium liquid, the positive hole of screening.Utilize limiting dilution assay or microclone method that positive hole is cloned, obtain the hybridoma cell strain of monoclonal antibody.
(4) cell cryopreservation and recovery: take the hybridoma frozen stock solution being in trophophase and make cell suspension, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen.Recovery room takes out cryopreservation tube, is immediately placed in 37 DEG C of water-baths instant, after centrifugal segregation frozen stock solution, moves into and cultivates culture in glassware.
(5) preparation of monoclonal antibody and purification: adopt and internal induce method, Balb/C week old mouse peritoneal is injected sterilizing paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomies, ascites is gathered after 7～10 days, after affinity chromatograph column purification, bottle subpackage ,-20 DEG C of preservations.
The preparation of benzo (a) pyrene polyclonal antibody: adopt new zealand white rabbit as immune animal, for immunogen, new zealand white rabbit is carried out immunity with benzo (a) pyrene and carrier protein couplet thing, repeatedly measure serum antibody titer after immunity, Culling heart blood, after affinity chromatograph column purification, obtain polyclonal antibody, bottle subpackage ,-20 DEG C of preservations.
Present invention also offers the using method of described benzo benzo (a) pyrene pyrene enzyme-linked immunologic detecting kit, comprise the following steps:
1, test kit is taken out from cold storage environment, balance to room temperature；
2, standard substance or (through pre-treatment) testing sample are added in the ELISA Plate hole being coated with also benzo (a) pyrene antigen, be subsequently added antibody working solution and ELIAS secondary antibody working solution, pat mixing, incubated at room；
3, wash with distilled water or ultra-pure water；
4, adding nitrite ion in every hole, pat mixing, room temperature lucifuge is hatched；
5, adding stop buffer, mix homogeneously in every hole, under 450nm wavelength, microplate reader reads light absorption value；
6, Analysis of test results, mean value calculation Relative Absorbance value with obtained standard substance light absorption value, with Relative Absorbance value for vertical coordinate, the semilog value of benzo (a) pyrene concentration is abscissa drawing standard curve, draw linear equation, calculate the Relative Absorbance value of sample solution by same method, obtain benzo (a) the pyrene concentration of counter sample according to equation.The calculating formula of described Relative Absorbance value is:
Relative Absorbance value (%)=(B/B0) × 100%
Wherein, B is the mean absorbance values of standard solution or sample solution, B0It it is the mean absorbance values of 0 concentration standard solution.The analysis of described testing result can also utilize computer professional software be calculated and analyze.
The equilibrium at room temperature that is placed in described in step 1 refers at room temperature (25～27 DEG C) placement more than 30min, makes the temperature of test kit recover to room temperature.
Described in step 2, pre-treating method is as follows:
Water sample:
Preparation method 1: take 10ml water sample, adjusts pH value to 6-8 with 0.1MHCL or 0.1MNaOH, takes 50 μ l and carry out ELISA detection after being mixed by water sample；Obtain the lowest detection of benzo (a) pyrene in water according to above-mentioned preparation method and be limited to 5 μ g/L.
Preparation method 2: take 10ml water sample, add isopyknic dichloromethane, static 3min after mixing, supernatant extracts once with isopyknic dichloromethane again, discard upper strata aqueous phase, merge the extracting solution of twice, after nitrogen dries up, redissolving in 500 μ l sample diluting liquids, every hole is taken out 50 μ l and is carried out ELISA detection；Cycles of concentration: 20, obtains the lowest detection of benzo (a) pyrene in water according to above-mentioned preparation method and is limited to 0.5 μ g/L.
Pedotheque: after taking the mixing of 100～200g soil, 4 degree of stored refrigerated；Before detection, soil mixing accurately being weighed 5g sample in 50ml centrifuge tube, add 25ml methanol, vortex oscillation 3～5min, 4000rpm are centrifuged 5min, take 50 μ l and carry out ELISA detection.
Compared with prior art, there is advantages that
Test kit provided by the invention adopts indirect competition ELSIA detection method, and the antibody specificity of employing is high, and affinity is strong, improves the sensitivity of detection, accuracy；Utilizing envelope antigen that ELISA Plate is coated, be coated compared to antibody, what the present invention was antigen coated is coated better effects if, and the holding time is longer, thus improve precision and the stability of test kit detection.
Additionally, no matter for water or soil sample, test kit provided by the invention is without complicated pretreatment process compared with traditional instrumental method, mensuration can be made directly through simply dealt sample, calculate the content of benzo (a) pyrene in sample according to the extension rate of computing formula and sample.
Test kit of the present invention have simple to operate quickly, stability is high, pin-point accuracy is sensitive, the advantages such as detection limit is low, and equipment and operator are required low, with low cost, it is highly suitable for trace analysis and the batch detection of the content of benzo (a) pyrene, it is suitable for the examination of a large amount of samples, solves the problem that main equipment is relied on by the detection of current benzo (a) pyrene, there is important application value.
Attached body explanation
Fig. 1 is benzo (a) pyrene canonical plotting
Detailed description of the invention
Below by specific embodiments, the present invention will be further described, but does not mean that limiting the scope of the invention.
Method described in following proposal, if no special instructions, is conventional method；Described reagent and material, if no special instructions, all can buy from commercial channel.
Example 1: prepared by antigen
Prepared by benzo (a) pyrene antigen
1, weigh 0.01mmolBAP derivant, 0.01mmolNHS, 0.01mmolDCC respectively, be dissolved in 4mlDMF, left at room temperature over night, 12000rpm, centrifugal 15min, separation of supernatant.
2, weigh 0.2gBSA to be dissolved in the sodium carbonate-bicarbonate buffer of 10ml0.05mol/L, pH9.6.
3, under 4 DEG C of conditions, the supernatant of preparation in step 1 is slowly added in solution described in 8ml step 2, continues stirring 5h
4, loading in bag filter by the reactant mixture of gained in step 3, after distilled water dialysis 2d, continue dialysis with pH7.4,0.01mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, every day changes dialysis solution.
5, every day above-mentioned dialysis solution is carried out absorption spectrum scanning, until can't detect the characteristic absorption peak of BaP
6, the dialysis solution in step with centrifugal separation 5, supernatant is immunogen BAP-BSA
Envelope antigen BAP-OVA is prepared according to above-mentioned 1～6 step same method
Example 2: prepared by antibody
Benzo (a) pyrene monoclonal antibody preparation
Animal immune program: adopting Balb/c mice as immune animal, benzo (a) the pyrene antigen of preparation is for immunogen in example 1, and immunizing dose is 60 μ g/, carried out a booster immunization afterwards every two weeks, and immunity amount is a 30 μ g albumen/mice.After 4th booster immunization, ELISA method detection titer, select two mices that titer is higher, immunizing dose is a 50 μ g albumen/mice, and immunity is impacted once in abdominal cavity, immunity one week after, takes mouse boosting cell.
Cell fusion and cloning: take immune mouse Balb/c splenocyte and SP2/0 cell, adopt PEG method to merge, and adopts indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, the positive hole of screening.Again screen after the positive cell strain screened is carried out sub-clone, until positive rate is 100%, finally give the hybridoma cell strain of stably excreting monoclonal antibody.
Cell cryopreservation and recovery: take the hybridoma frozen stock solution being in trophophase and make cell suspension, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen.Recovery room takes out cryopreservation tube, is immediately placed in 37 DEG C of water-baths instant, after centrifugal segregation frozen stock solution, moves into and cultivates culture in glassware.
The preparation of monoclonal antibody and purification: adopt and internal induce method, inject sterilizing paraffin oil by Balb/C week old mouse peritoneal, 7～14 days pneumoretroperitoneum injection hybridomies, gathers ascites after 7～10 days, after affinity chromatograph column purification, and bottle subpackage ,-20 DEG C of preservations.
Example 3: the preparation of enzyme linked immunological kit component
(1) being coated of ELISA Plate: antigen pH9.6, the carbonate buffer solution of 0.05mol/L is diluted to 0.5～1ug/ml, wherein carbonate buffer solution is containing 0.1～0.2% sodium carbonate and 0.2%～0.3% sodium bicarbonate, every hole of ELISA Plate adds 100 μ l, 4 DEG C are coated overnight or 37 DEG C and are coated 2h, incline and be coated liquid milli-Q water 3 times, pat dry in every hole, then add 100 μ l confining liquids, put in 37 DEG C of incubators and use milli-Q water 3 times after 2h, in dried inclosure aluminium foil bag, 4 DEG C of preservations.
(2) confining liquid: the phosphate buffer of the pH7.4 containing 2%BSA, wherein phosphate buffer Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
(3) benzo (a) pyrene standard solution: accurately weigh benzo (a) pyrene standard specimen 10mg, be dissolved in 10ml methanol, then ultra-pure water is diluted to subpackage 4 DEG C preservation after 10 μ g/ml.During use, a series of benzo (a) the pyrene standard solutions becoming concentration to be 0.5 μ g/ml, 0.1 μ g/ml, 0.02 μ g/ml, 0.004 μ g/ml standard solution stepwise dilution with ultra-pure water.
(4) sample diluting liquid: for containing 30% methanol, 1%～5% bovine serum albumin and 0.05% polysorbas20, the phosphate buffer of pH7.4, wherein phosphate buffer Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
(5) antibody diluent: for containing 1% bovine serum albumin and 0.01% polysorbas20, the phosphate buffer of pH7.4, wherein phosphate buffer Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
(6) benzo (a) pyrene antibody working solution: benzo (a) pyrene monoclonal antibody is that laboratory early stage prepares, and sees example 2: prepared by antibody；Subpackage after by above-mentioned diluted to working concentration.
(7) ELIAS secondary antibody: the sheep anti mouse two for horseradish peroxidase-labeled is anti-or sample anti-rabbit two is anti-, commercialization reagent.
(8) nitrite ion: the commercialization one-component nitrite ion containing TMB (TMB).
(9) stop buffer: be 1～2mol/L sulfuric acid solution.
(10) reagent subpackage: various reagent are prepared on request, measures qualified rear aseptic subpackaged.Antibody diluent, nitrite ion, benzo (a) pyrene antibody working solution are 12ml/ bottle；Sample diluting liquid, stop buffer are 10ml/ bottle；Benzo (a) pyrene standard solution 1ml/ bottle；ELIAS secondary antibody is 150ul/ pipe.Label after subpackage dated lot number and effect duration, 4 DEG C of preservations.
(11) test kit assembles: will detachably be coated the microwell plate 1 piece of antigen respectively, benzo (a) pyrene antibody working solution, ELIAS secondary antibody, sample diluting liquid, benzo (a) pyrene standard solution, antibody diluent, nitrite ion and each one bottle of stop buffer/pipe, operation instructions are a, specify position to test kit, test kit encapsulates after the assay was approved, 4 DEG C of preservations.
Example 4: set up the enzyme linked immunological kit of detection benzo (a) pyrene, comprise following component:
(1) 96 hole ELISA Plate of benzo (a) pyrene antigen it are coated, 12 × 8/ pieces, 1 piece
(2) benzo (a) pyrene antibody working solution, 12ml/ bottle, 1 bottle
(3) enzyme labelling two resists, 150 μ l/ pipes, 1 pipe
(4) benzo (a) pyrene standard solution, concentration is 0ppb, 4ppb, 20ppb, 100ppb, 500ppb, 1ml/ bottle, 1 bottle
(5) sample diluting liquid, 10ml/ bottle, 1 bottle
(7) antibody diluent, 12ml/ bottle, 1 bottle
(8) nitrite ion, 12ml/ bottle, 1 bottle
(9) stop buffer, 10ml/ bottle, 1 bottle
(12) operation instructions, 1 part
(14) valve bag (containing desiccant), 1
Example 5: testing sample pre-treatment
1, water sample:
Preparation method 1: take 10ml water sample, adjusts pH value to 6-8 with 0.1MHCL or 0.1MNaOH, takes 50 μ l and carry out ELISA detection after being mixed by water sample；Obtain the lowest detection of benzo (a) pyrene in water according to above-mentioned preparation method and be limited to 5 μ g/L.
Preparation method 2: take 10ml water sample, add isopyknic dichloromethane, static 3min after mixing, supernatant extracts once with isopyknic dichloromethane again, discard upper strata aqueous phase, merge the extracting solution of twice, after nitrogen dries up, redissolving in 500 μ l sample diluting liquids, every hole is taken out 50 μ l and is carried out ELISA detection；Cycles of concentration: 20, obtains the lowest detection of benzo (a) pyrene in water according to above-mentioned preparation method and is limited to 0.5 μ g/L.
2, pedotheque: after taking the mixing of 100～200g soil, 4 degree of stored refrigerated；Before detection, soil mixing accurately being weighed 5g sample in 50ml centrifuge tube, add 25ml methanol, vortex oscillation 3～5min, 4000rpm are centrifuged 5min, take 50 μ l and carry out ELISA detection.
Example 6: use the method that test kit carries out detecting
1, being taken out from cold storage environment by test kit, be placed in room temperature (25～27 DEG C) balance more than 30min, be fixed on sheet frame by the lath of quantity used by enough standard substance and sample, standard substance and sample and Quality Control do two parallel laboratory tests, number in order.
2, prepared by ELIAS secondary antibody working solution: 1 volume two is anti-with 99 volume antibody diluents mixing (100 times of dilutions)
3, the standard substance of 50 μ l are added in set hole；
4, the sample of 50 μ l is added in set hole
5, in every hole, add 100 μ l primary antibodie working solutions, the two anti-working solutions of 100 μ l, rap microwell plate edge and mix 1 minute；
Room temperature (25 ± 2) DEG C lucifuge hatches 30 minutes；
6, wash plate 2 times, 300ul distilled water or deionized water should be added every time, make plate dry as far as possible and blot in absorbent paper for the last time；
7, the nitrite ion of 100 μ l is added；Timing, mixes one minute；
8, hatching 15min in room temperature (25 ± 2) DEG C lucifuge, the stop buffer adding 50 μ l terminates reaction,
9, under 450nm and 630nm dual wavelength, read OD value (before reading, remove the moisture bottom micropore with lint-free cloth wiping and refer to trace)
10, testing result calculates and analyzes:
With the mean value calculation Relative Absorbance value of obtained standard solution absorbance, with Relative Absorbance value for vertical coordinate, the semilog of benzo (a) pyrene concentration of standard solution is abscissa drawing standard curve, obtains linear equation.Calculate the Relative Absorbance value of sample solution by same method, obtain benzo (a) the pyrene concentration in corresponding each sample well according to equation, then be multiplied by corresponding extension rate, both obtain the actual concentrations of benzo (a) pyrene in sample.
Described Relative Absorbance value (%)=(B/B0)×100
Wherein, B is the mean absorbance values of standard solution or sample, B0It it is the mean absorbance values of 0 μ g/L standard solution.
Example 7 kit standard curve determination
Benzo (a) pyrene standard solution is utilized to react, according to experimental result drawing standard curve, see accompanying drawing 1, linear equation is y=-0.383x+0.0806, the logarithm value of Relative Absorbance value (%) and benzo (a) pyrene concentration is in significant linear relationship within the scope of concentration 0.004～0.5mg/L, and correlation coefficient is R2=0.9916, the sensitivity of test kit is 0.005mg/L, is limited to 5 μ g/L to without the lowest detection of water extracts.
Example 8 test kit preci-sion and accuracy is tested
Accuracy refers to the matching degree of measured value and true value, and in ELISA measures, accuracy represents with TIANZHU XINGNAO Capsul, and precision represents with the coefficient of variation (CV).Tap water sample without benzo (a) pyrene adds benzo (a) the pyrene standard solution of final concentration of 5 μ g/L, 10 μ g/L, 20 μ g/L, each batch every kind add concentration do 8 parallel, calculate TIANZHU XINGNAO Capsul meansigma methods and CV value.
Tap water sample TIANZHU XINGNAO Capsul measurement result is in Table 1
It is shown that the average recovery rate of above 3 pitch-based sphere ranges for 70.0%～106.1%, variation within batch coefficient is 3.5%～8.6%.
The comparison of water sample detection and standard method
This kit method and GB/T5750.8-2006 (drinking water standard method of inspection organic indicator) compare, and the sample adding concentration known is analyzed.Tap water sample without benzo (a) pyrene adds benzo (a) the pyrene standard solution of final concentration of 5 μ g/L, 10 μ g/L, 20 μ g/L.
Test kit and standard method comparative result are in Table 2.
The comparison of Soil K+adsorption and standard method
This kit method and EPA8270D gas chromatograph-mass spectrometer (GC-MS) measure semi-volatile organic compounds (GC/MS) and compare, actual contaminated soil sample is analyzed, selects the laboratory of two different testing agencies to carry out the detection of Instrumental Analysis (GC/MS) method respectively.Result is in Table 3.
The test kit testing result of tap water and soil sample is basically identical with national standard method and EPA standard method testing result.
In sum, enzyme-linked immunologic detecting kit of the present invention has extraordinary preci-sion and accuracy, and meets country's prescription to all kinds of index of test kit.
Example 9 test kit storage life is tested
1, test kit is positioned over 2～8 DEG C, take the test kit behind 0,2,4,6,8,9,10,11 and 12 months respectively, absorbance to benzo (a) pyrene standard substance (0.005mg/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter such as variation within batch coefficient is measured.
2, by test kit 37 DEG C preserve when place 12 days, every day the absorbance to benzo (a) pyrene standard sample (0.005mg/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient are measured.
3, by test kit-20 DEG C of Refrigerator stores 12 days, every day the absorbance to benzo (a) pyrene standard sample (0.005mg/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch coefficient are measured.
4, result shows, through three kinds of condition food preservation test, the absorbance of benzo (a) pyrene standard substance (0.005mg/L) is dropped by less than 5%, 50% suppression ratio is between 0.05～0.2mg/L, TIANZHU XINGNAO Capsul is between 70～110%, and variation within batch coefficient is less than 10%, and indices conforms to quality requirements, therefore, test kit of the present invention can preserve December at 2～8 DEG C.
Claims (10)
1. the enzyme-linked immunologic detecting kit of detection benzo (a) pyrene, it is characterised in that described test kit includes following component:
(1) ELISA Plate (96 hole) of benzo (a) pyrene antigen it is coated with；
(2) benzo (a) pyrene specific antibody；
(3) benzo (a) pyrene standard solution；
(4) ELIAS secondary antibody；
(5) sample diluting liquid；
(6) antibody diluent；
(7) nitrite ion；
(8) stop buffer.
2. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that described benzo (a) pyrene antigen is obtained by activity fat method coupling by benzo (a) pyrene hapten and carrier protein, described carrier protein is bovine serum albumin or oralbumin.
3. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that described sample diluting liquid is for containing 30% methanol, 1%～5% bovine serum albumin and 0.05% polysorbas20, the phosphate buffer of pH7.4, wherein phosphate buffer Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
4. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that described antibody diluent is for containing 1% bovine serum albumin and 0.01% polysorbas20, the phosphate buffer of pH7.4, wherein phosphate buffer Contents of Main Components is KH2PO40.2%, Na2HPO43%, NaCl8%, KCl0.2%.
5. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterised in that described benzo (a) pyrene specific antibody is the monoclonal antibody for preparing of the immunization method routinely of benzo (a) pyrene antigen described in claim 2 or polyclonal antibody.Its working solution is that specific antibody mixes according to the ratio of 1: 5000～10000 with above-mentioned antibody diluent.
6. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterized in that described ELIAS secondary antibody is sheep anti mouse or goat anti-rabbit antibody, working solution used is that enzyme labelled antibody mixes according to the ratio of 1: 100～200 with above-mentioned antibody diluent.
7. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterised in that described benzo (a) pyrene standard solution concentration is the series of standards product solution of 0ppb, 4ppb, 20ppb, 100ppb, 500ppb.
8. benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 1, it is characterised in that described stop buffer is 1～2mol/L sulfuric acid solution.
9. claim 1～8 arbitrary described benzo (a) the pyrene enzyme linked immunological kit using method when detecting benzo (a) pyrene residual, it is characterised in that comprise the steps:
(1) test kit is taken out from cold storage environment, balance to room temperature；
(2) standard substance or add through the testing sample of pre-treatment have been coated with in the ELISA Plate hole of benzo (a) pyrene antigen, have sequentially added antibody working solution and ELIAS secondary antibody working solution, pat mixing, incubated at room；
(3) wash with distilled water or deionized water；
(4) adding nitrite ion in every hole, pat mixing, room temperature lucifuge is hatched；
(5) adding stop buffer, mix homogeneously in every hole, under 450nm wavelength, microplate reader reads light absorption value；
(6) Analysis of test results, according to standard concentration and relative light absorption value drawing standard curve, calculates sample concentration in well, it is determined that benzo (a) pyrene content in actual sample.
10. the using method of benzo (a) pyrene enzyme-linked immunologic detecting kit according to claim 9, it is characterized in that, the pre-treatment described in (2) refers to that the relatively low water sample of levels and pedotheque need just to can be used for detecting through pretreatment process.
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| CN115028732B (en) * | 2022-07-11 | 2023-09-08 | 华中农业大学 | Anti-benzo [ a ] pyrene monoclonal antibody and application thereof |
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