CN104711230B - Hybridoma DBP-4E10 and its dibutyl phthalate monoclonal antibody of generation - Google Patents
Hybridoma DBP-4E10 and its dibutyl phthalate monoclonal antibody of generation Download PDFInfo
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Abstract
The anti-dibutyl phthalate DBP monoclonal antibodies generated the present invention provides hybridoma cell strain 4E10, its secretion and its application.Hybridoma cell strain 4E10 can be used for preparing high-titer dibutyl phthalate DBP antibody, and the potency that mouse hydroperitoneum antibody of RGDV Enzyme Linked Immunoadsorbent Assay (ELISA) method measures is up to 3.12 × 106；The anti-dibutyl phthalate DBP monoclonal antibody high sensitivities, 50% inhibition concentration, that is, IC50 to dibutyl phthalate DBP is 0.22 mg/mL, is used to measure dibutyl phthalate DBP contents, actual application value is big.
Description
Technical field
The present invention relates to hybridoma cell strain DBP-4E10 and its dibutyl phthalate monoclonal antibodies of generation.
Background technology
Phthalate compound (PAEs) is a kind of environmental hormone substance, has estrogenic activity, interference life
Object internal system normal function leads to mutation, distortion and cancer cell multiplication.2005, European Union was pungent by phthalic acid two
Ester (DEHP), dibutyl phthalate (DBP) and BBP(Butyl Benzyl Phthalate (BBP) are assessed as " the second class genotoxicity ".
Wherein, DBP is considered as the highest phthalate compound of toxicity.
PAEs compounds are the main plasticizer and softening agent of plastics industry, be widely used in packaging material for food, container,
The manufacture of medical supplies and plastics etc..Due to its extensive use and from the release in various products, phthalate compound is
Through each corner for almost penetrating into our lives.Studies have shown that China's resident's DBP daily intakes are 12.2mg/kg, surpass
Cross the daily maximum permissible intake standard of the 10mg/kg.d of World Health Organization's suggestion.Food is considered as that the mankind are exposed to
The main path of phthalate compound.In food the pollution of phthalate compound mostly come from the plastics package of food with
And production process.This is because phthalate ester is a kind of fat-soluble compound, it is easily entered in the fat of food, grease, and
It is enriched with wherein.
Existing detection method GC, MS, HPLC measurement DBP high sensitivities of DBP, accuracy are good, and can measure knot respectively
The similar multiple compounds of structure and its isomers are current optimal detection methods.But this kind of analysis method system will wait for
Survey pollutant from sample separation and Extraction and remove impurity interference pretreatment process it is sufficiently complex, when operating cost, analysis cost
It is expensive.So easy to operate, quick, economic and high sensitivity the detection method of exploitation has important practical significance.
The high specific of immune response and modern means of testing high sensitivity are combined by immunoassay method, are had fast
Speed, economy, the advantages that sample aequum is few, pre-treatment is simple, become modern biochemistry, medicine, chemical analysis and environment prison
The research hotspot in the fields such as survey.Immunoassay is the mark on association reaction and antigen, antibody using the specificity of antigen-antibody
The amplification of note object to carry out qualitative and quantitative analysis to ultramicron determinand, so to study foundation is directed to phthalic acid
Any type immunology detection technology of dibutyl ester (DBP), it is necessary to first obtain the anti-of anti-dibutyl phthalate (DBP)
Body.
Invention content
Problem to be solved by this invention is to provide hybridoma cell strain DBP-4E10 and its phthalic acid two of generation
The monoclonal antibody of butyl ester DBP
The present invention provides the thin strain DBP-4E10 of hybridoma, it is micro- which has been preserved in China on December 12nd, 2014
Biological inoculum preservation administration committee common micro-organisms center (CGMCC), preservation address are China, the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC NO.10202.Classification And Nomenclature is S/P20
Hybridoma.
Anti- dibutyl phthalate monoclonal antibody is by the hybridoma cell strain that deposit number is CGMCC NO.10202
DBP-4E10 secretions generate.The DBP monoclonal antibodies can identify dibutyl phthalate, to dibutyl phthalate
50% inhibition concentration IC50 is 0.22mg/L.
Application of the anti-dibutyl phthalate monoclonal antibody in dibutyl phthalate measurement.
Hybridoma cell strain DBP-4E10 monoclonal antibodies provided by the invention are screened using three-step approach and are obtained, tool
Body step is：With 4- nitrophthalic acid acid anhydrides in H2SO4Effect is lower and n-butanol reaction generates two fourth of 4- nitrophthalic acids
Then nitro is reduced into amino by ester, diazotization and then and BSA occur in the presence of finally in hydrochloric acid and sodium nitrite
Coupling generates DBP-BSA.BLAB/c mouse are immunized 4 times with it, last time booster immunization 2 times and a preceding immunizing dose
DBP-BSA carries out cell fusion after 3 days immune.Using ELISA, method carries out screening fused cell in two steps：Between the first step uses
It connects ELISA method and filters out the positive colony for being only resistant to dibutyl phthalate without anti-carrier protein BSA；Between second step uses
It connects Inhibition ELISA to be detected the positive colony culture solution that the first step filters out, using DBP as competition source, selection is inhaled
Shading value is low, positive colony in higher sensitivity carries out limiting dilution assay and continues to clone, and clone uses same two step after 10 days
Screening method carries out antibody test, is so repeated 2 times, finally screens the hybridoma DBP-4E10 of acquisition.
The preparation method of dibutyl phthalate monoclonal antibody provided by the invention, steps are as follows：By the miscellaneous of acquisition
It hands over tumor cell strain DBP-4E10 injections to use the processed BALB/c mouse of incomplete Freund's adjuvant in advance, collects the abdomen of the mouse
Water obtains the monoclonal antibody of anti-dibutyl phthalate after purification process.
The concrete operations of above-mentioned purification process are：For ascites in 4 DEG C, 3000r/min centrifuges 5min or more, draws supernatant, will
Supernatant is mixed with the acetate buffer of 2 times of volumes, is slowly added to caprylic acid, the caprylic acid needed for every milliliter of ascites while stirring
Volume is 33 μ L, and mixed at room temperature 30min, 4 DEG C of standing 2h or more make it fully precipitate.Then 4 DEG C, 12000r/min centrifugations
30min or more abandons precipitation, and after obtained supernatant is filtered with sand core funnel, the 0.1mol/L of 1/10 filtrate volume is added
The phosphate buffer of pH7.4 adjusts pH to 7.4 with the sodium hydroxide solution of 2mol/L, and 4 DEG C are pre-chilled 1 hour, are slowly added to
For 0.277g/mL ammonium sulfate to ammonium sulfate final concentration of 45%, 4 DEG C stand 2h or more, and then 4 DEG C of 12000r/min centrifuge 30min,
Supernatant is abandoned, gained is precipitated and is resuspended with the 0.01mol/L phosphate buffers of former ascites volume 1/10, bag filter is packed into, is used
0.01mol/L phosphate buffers are dialysed, and isometric glycerine is added in the protein solution fully dialysed, is set in -20 DEG C of refrigerators
It is spare；
Acetate buffer：0.141mL acetic acid is added in 0.29g sodium acetates, and pure water is settled to 100mL；
0.1mol/L phosphate buffers：0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride,
0.02g potassium dihydrogen phosphates, add water to be settled to 100mL.
0.01mol/L phosphate buffers：0.1mol/L phosphate buffers are diluted 10 times with pure water.
The beneficial effects of the present invention are：
1 hybridoma DBP-4E10 provided by the invention can be used for preparing the anti-dibutyl phthalate of high-titer
Monoclonal antibody, the potency that dibutyl phthalate mouse ascites antibody ELISA immuno absorbence method measures is reached 3.12 ×
106, i.e. ascites is diluted to 3.12 × 106When solution measurement result be still the positive.
2 dibutyl phthalate monoclonal antibody high sensitivities provided by the invention, specificity are good, to phthalic acid
The 503nhibiting concentration IC50 of dibutyl ester is 0.22mg/L, the cross reacting rate 36.9% with diisobutyl phthalate, with neighbour
The cross reacting rate 10.7% of phthalic acid butyl benzyl is respectively less than 1% with the cross reacting rate of other structures analog.
The monoclonal antibody of 3 anti-dibutyl phthalates provided by the invention can be used for measuring dibutyl phthalate
Content.
Description of the drawings
Standard curve of Fig. 1 DBP-4E10 monoclonal antibodies to DBP；
Specific implementation mode
Embodiment 1：The screening of hybridoma cell strain DBP-4E10
1. the synthesis of antigen and being immunized for animal
Esterification occurs under 4- nitrophthalic acids acid anhydride and n-butanol carded sliver part, obtains 4- nitrophthalic acids two
Butyl ester, then reduction reaction occurs with zinc powder in acid condition, obtain hapten derivant 4- aminophthalic acid dibutyl esters
Ester has the phthalic acid ester hapten derivant of phenylamino that diazo-reaction occurs with NaNO2 and dense HCl at low temperature, raw
At corresponding diazol；With carrier protein coupling reaction occurs for diazol, obtains corresponding artificial antigen.
The synthesis of 4- nitrophthalic acid dibutyl esters
2.5 grams of 4- nitrophthalic acids are dissolved in 50mL n-butanols, the dense H2SO4 of 1mL is slowly dropped into thereto and urges
Agent is reacted and is stirred continuously at 120 DEG C.After reaction, it is washed three times with 10%NaCO3, each 10mL.80 DEG C of rotations
Evaporation removes n-butanol and water.Oily mater 4- nitrophthalic acid dibutyl esters are obtained, are weighed.
The synthesis of 4- aminophthalic acid dibutyl ester esters
Weigh 0.1523 gram of 4- nitrophthalic acid dibutyl ester；28mL benzene and 0.28g zinc powders is added, then adds in three times
Enter the dense HCl of 8.2mL, 0.28 gram of zinc powder is added after being stirred to react 18min, reaction 12h is stirred at room temperature.After reaction, liquid separation；
Then benzene layer is cleaned with 34mLH2O, uses 5g anhydrous Nas 2SO4 dryings benzene layer respectively three times, 50 DEG C of rotary evaporations remove benzene.It obtains yellow
Color 4- aminophthalic acid dibutyl ester solids, weigh.
The synthesis of artificial antigen
Configure A liquid：0.0466 gram of 4- aminophthalic acids dibutyl ester is weighed in 50mL triangular flasks, is sequentially added
Dense HCl, 3mLH2O and 2mL dimethylformamides of 0.1mL.It sets under 5 degree of ice bath, stirs.0.2mol/L sodium nitrites are added dropwise,
Become blue to starch KI test paper, reacts 30 minutes.
Configure B liquid：Sodium borate buffer solution (pH9.0) 40mL of 4mg/mLBSA.
B liquid is added dropwise in A liquid, gradually there is orange-red solution appearance, the reaction was continued 2h obtains crude product.Reaction solution fills
Enter in bag filter, phosphate buffer (pH7.40.01mol/L) is dialysed 3 days, changes dialyzate twice daily.It collects in bag filter
Liquid, DBP-BSA artificial antigens as obtained.
The identification of artificial antigen
Show the comlete antigen DBP- that dibutyl phthalate has been prepared by the identification of uv scan method
BSA。
6 week old female BAl BIcs/c mouse 6 are bought, the comlete antigen of the immune dibutyl phthalate voluntarily synthesized
DBP‐BSA.It is immune for the first time by comlete antigen DBP-BSA and equivalent Freund's complete adjuvant mixing and emulsifying, then mouse nape part
Subcutaneous multi-point injection.It is immune to after head exempts from 3 weeks and carries out for the second time, using incomplete Freund's adjuvant and isometric comlete antigen DBP-
BSA is emulsified, and immunizing dose is exempted from method with head identical.Third time is immune with second of 3 weeks immunization interval time, immunizing dose with
Method is identical as second.Be immune to for 4th time third time it is 3 weeks immune after carry out, immunization ways and immunizing dose with exempt from for the third time
Epidemic disease is identical, and each immunizing dose is per 50 μ g of mouse.Latter week is 3 times immunized every time, eye circumference blood sampling detaches serum, using indirect
ELISA method monitors mice serum antibody titer.3 days after the 4th is immune, tail vein blood detaches serum, using indirect ELISA
Method monitors mice serum antibody titer, and indirect competitive ELISA method is used in combination to measure mice serum sensitivity, selects potency, sensitivity
The corresponding mouse of relatively higher serum carries out last time booster immunization, immunizing dose be before 2 times.2. cell fusion
After last time booster immunization 3 days, using the polyethylene glycol (PEG, molecular weight 1460) of 50% (weight percent)
Make fusion agent, carries out cell fusion according to a conventional method.It is as follows：
BALB/c mouse is taken off neck to put to death, is soaked in 75% alcohol.It is put into super-clean bench after about 3min.Lifted with tweezers small
Mouse skin of abdomen cuts an osculum (attention does not break peritonaeum) with operating scissors, then tears skin to head, tail both direction with tweezers
Skin fully exposes peritonaeum.It uses clean scissors instead and cuts off peritonaeum, take out spleen, be put into and filled the flat of the endless full nutrient solutions of 10mL
It is cleaned once in ware, and peels off connective tissue.Spleen is transferred in another plate for filling the endless full nutrient solutions of 5mL, a bronze medal is taken
Spleen is placed on copper mesh by net, and spleen is ground with plunger, is used in combination the endless full nutrient solution in plate to rinse copper mesh, is made
Splenocyte is all entered by mesh in solution.Spleen cell solutions are transferred in 50ml centrifuge tubes, add endless full nutrient solution extremely
30mL, mixing.1000rpm centrifuges 5min, abandons supernatant.Sedimentation cell cannots be used up full nutrient solution centrifugation once again, then by cell
It is suspended from 10ml complete culture solutions, mixing.Cell count, it is spare.
Myeloma cell and splenocyte are pressed 1：10 ratio mixing, is washed 1 time in 50mL centrifuge tubes with DMEM culture mediums,
1200rpm centrifuges 8min；Supernatant is abandoned, with suction pipe exhaustion residual liquid.It is merged with 50%PEG, with containing 20% cow's serum, 1%HAT
DMEM culture mediums are resuspended.It is added to above-mentioned cell in 96 orifice plates of existing feeder layer, adds 100 μ L per hole.Culture plate is set
37 DEG C, cultivate in 5%CO2 incubators.DMEM culture mediums are the bases the DMEM culture containing 2% (weight percent) growth factor
Base, DMEM basal mediums are purchased from Hyclone companies, and (hypoxanthine-aminopterin-thymidine is purchased from 1%HAT
Gibco companies.
3. screening and the clone of cell strain
The 10th day or so after cell fusion, cell colony, which is grown to, accounts for 1/2 size of bottom hole, you can carries out antibody inspection
It surveys.Using ELISA method to there is the culture hole of Growth of Hybridoma Cell to screen, screening is carried out in two steps, and the first step uses
Indirect elisa method filters out positive hole of the anti-dibutyl phthalate without anti-carrier protein BSA；Second step is using indirectly competing
It strives ELISA method to be detected the positive hole that the first step filters out, uses dibutyl phthalate former as competition, select extinction
Value and sensitivity higher hole (light absorption value is higher refer to competition be originally 0 the hole i.e. final tested volume of negative control hole it is higher, spirit
Competition original content that is, 50 values of IC when the higher finger inhibiting rate of sensitivity is 50% is smaller), it is using limiting dilution assay that hybridoma is thin
Born of the same parents' cloning is detected for 10 days or so after clone using same two-step method, after such repeated cloning 2-3 times, is hybridized
Tumor cell strain DBP-4E10.
Embodiment 2：Preparation, purifying and the identification of antibody
The hybridoma cell strain DBP-4E10 of acquisition is injected in advance at incomplete Freund's adjuvant by what embodiment 1 obtained
The BALB/c mouse managed collects the ascites of the mouse, and the monoclonal that anti-dibutyl phthalate is obtained after purification process is anti-
Body.
Concrete operations are：Ascites centrifuges 5min or more in 4 DEG C of 3000r/min, draws supernatant, by supernatant and 2 times of volumes
Acetate buffer mixes, and is slowly added to caprylic acid while stirring, and the caprylic acid volume needed for every milliliter of ascites is 33 μ L, room temperature
30min is mixed, 4 DEG C of standing 2h make it fully precipitate.Then 4 DEG C of 12000r/min centrifuge 30min, abandon precipitation, upper by what is obtained
After clear liquid is filtered with sand core funnel, the phosphate buffer of the 0.1mol/L pH7.4 of 1/10 filtrate volume is added, uses 2mol/L
Sodium hydroxide solution adjust pH to 7.4,4 DEG C be pre-chilled 1 hour, be slowly added to 0.277g/mL ammonium sulfate to ammonium sulfate final concentration
It is 45%, 4 DEG C of standing 2h, 4 DEG C of 12000r/min centrifuge 30min, abandon supernatant, by gained precipitation former ascites volume 1/10
0.01mol/L phosphate buffers are resuspended, and are packed into bag filter, are dialysed, will fully be dialysed with 0.01mol/L phosphate buffers
Protein solution isometric glycerine is added, set spare in -20 DEG C of refrigerators；
Acetate buffer：0.141mL acetic acid is added in 0.29g sodium acetates, and pure water is settled to 100mL；
0.1mol/L phosphate buffers：0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride,
0.02g potassium dihydrogen phosphates, add water to be settled to 100mL.
0.01mol/L phosphate buffers：0.1mol/L phosphate buffers are diluted 10 times with pure water.
It is reachable with the potency for the mouse hydroperitoneum antibody of RGDV that routinely non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method measures 2D10
3.12×106, i.e. ascites is diluted to 3.12 × 106When measurement result be the positive.Identify that its is right with conventional indirect competitive ELISA method
503nhibiting concentration IC50 to dibutyl phthalate is 0.22mg/L, the cross reacting rate with diisobutyl phthalate
36.9%, the cross reacting rate 10.7% with BBP(Butyl Benzyl Phthalate is small with the cross reacting rate of other structures analog
In 1%.
Embodiment 3：The application of antibody
The anti-dibutyl phthalate monoclonal antibody that hybridoma cell strain DBP-4E10 is secreted is used for O-phthalic
Dibutyl phthalate monoclonal ELISA adds recovery test, is as follows：
(1) it is coated with：Using 96 hole polystyrene ELISA Plates, with the carbonic acid buffer of 0.05mol/L pH 9.6 by DBP-
BSA is diluted to 1 μ g/mL, 100 μ L is added into per hole with the adjustable liquid-transfering gun of multiple tracks (the abbreviation volley of rifle fire), by ELISA Plate after adding
It is covered with preservative film, is placed in 4 DEG C overnight.
(2) board-washing：Second day, ELISA Plate is taken out from 4 DEG C, is washed using PBST, per 250 μ l of hole, washs 4 times, washes
It is patted dry on towel afterwards.
(3) it closes：The PBS of 200 μ L 1%BSA is added into per hole with the volley of rifle fire, sets ELISA Plate with covering after adding
2h is placed in 37 DEG C.It is washed using PBST, per 250 μ l of hole, washs 4 times, patted dry on towel after washing.
(4) the dibutyl phthalate standard of 0,0.03,0.09,0.27,0.81,2.43mg/L are prepared with 35% methanol
Solution.Standard solution and detected sample extracting solution are separately added into the ELISA Plate closed, 50 holes μ L/, each sample
3 repeating holes of product, add and are diluted to 1：50000 dibutyl phthalate monoclonal antibody, 50 holes μ L/, add and are diluted to
1：40min is gently reacted in 3000 50 holes μ L/ of ELIAS secondary antibody after beating ELISA Plate in 37 DEG C of insulating boxs.
(5) board-washing：Operation is same as above.
(6) it develops the color：Developing solution A and developing solution B is pressed 1：1 volume ratio mixing, the developing solution mixed is added with the volley of rifle fire
37 DEG C of insulating box light protected environment reaction 10min are set in 100 holes μ L/.
(7) it measures：2mol/L H are added using the volley of rifle fire2SO450 holes μ L/, are gently vibrated ELISA Plate, are detected using microplate reader
OD values at 450/630nm wavelength.
(8) TIANZHU XINGNAO Capsul and sample pre-treatments：It weighs 5g white wine to be placed in clean 10mL teat glass, add respectively
1.5,3,5 μ g DBP are added the n-hexane of 2mL chromatographically pures, 3min are vibrated after closing the lid, and stand, supernatant liquor is taken after being layered
1mL is volatilized in clean glassware.It is molten with the methanol of 1mL 35% weight after volatilizing, it is transferred to conduct after 1.5ml centrifuge tubes
ELISA sample extracting solutions are to be measured.Rate of recovery experiment is added using indirect competitive ELISA, the rate of recovery is respectively 78.1,
89.9,109.3%.
(9) configuration of solution：
Carbonic acid buffer：Weigh Na2CO31.59g NaHCO32.93g is added pure water to 990mL, adjusts pH to 9.6, then use
Pure water is settled to 1000mL, and 4 DEG C of storages are spare.
Phosphate buffer (PBS)：8.5g NaCl, 2.2g Na2HPO4·12H2O, 0.2g NaH2PO4·2H2O is dissolved in
In 900mL pure water, pH to 7.4 is adjusted, 1000mL is settled to.
PBST：500mL PBS are taken, 0.25mL polysorbas20s are added, mixing is spare.
Developing solution A：20mg TMB are dissolved in 10mL DMSO, and 4 DEG C of preservations take out restore to room temperature before use, dilute with pure water
It releases to 100mL.
Developing solution B：21g monohydrate potassiums, 71.1g Na2HPO4·12H2O, 2g hydrogen peroxide urea add pure water constant volume
To 1000mL.
ELIAS secondary antibody is purchased from SoutherBiotech companies.
Claims (3)
1. hybridoma cell strain DBP-4E10, it is characterised in that：It has been preserved in China General Microbiological culture presevation administrative center,
Deposit number is CGMCC NO.10202, wherein the hybridoma will be produced using after DBP-BSA mice immunized with antigen
The lymphocyte of raw antibody merges screening with myeloma cell and obtains, wherein the preparation method of the DBP-BSA antigens is as follows：
1) synthesis of 4- nitrophthalic acids dibutyl ester
2.5 grams of 4- nitrophthalic acids are dissolved in 50mL n-butanols, are slowly dropped into the dense H of 1mL thereto2SO4Make catalyst,
It reacts and is stirred continuously at 120 DEG C；After reaction, 10%Na is used2 CO3It washs three times, each 10mL；80 DEG C of rotary evaporations
Remove n-butanol and water；Oily mater 4- nitrophthalic acid dibutyl esters are obtained, are weighed；
2) synthesis of 4- aminophthalic acids dibutyl ester
Weigh 0.1523 gram of 4- nitrophthalic acid dibutyl ester；28mL benzene and 0.28g zinc powders is added, is then added in three times
The dense HCl of 8.2mL add 0.28 gram of zinc powder after being stirred to react 18min, and reaction 12h is stirred at room temperature；After reaction, liquid separation；So
After use 34mLH2O cleans benzene layer, uses 5g anhydrous Nas respectively2SO4Three times, 50 DEG C of rotary evaporations remove benzene to dry benzene layer；Obtain yellow 4-
Aminophthalic acid dibutyl ester solid, weighs；
3) synthesis of artificial antigen
Configure A liquid：0.0466 gram of 4- aminophthalic acids dibutyl ester is weighed in 50mL triangular flasks, it is dense to sequentially add 0.1mL
HCl、3mLH2O and 2mL dimethylformamides；It sets under 5 degree of ice bath, stirs；0.2mol/L sodium nitrites are added dropwise, until starch
KI test paper becomes blue, reacts 30 minutes；
Configure B liquid：The sodium borate buffer solution 40mL, pH9.0 of 4mg/mL BSA；
B liquid is added dropwise in A liquid, gradually there is orange-red solution appearance, the reaction was continued 2h obtains crude product；Reaction solution is packed into saturating
It analyses in bag, using pH7.4, the phosphate buffer of 0.01M is dialysed 3 days, changes dialyzate twice daily；Collect the liquid in bag filter
Body, DBP-BSA artificial antigens as obtained；
4) identification of artificial antigen
Show the comlete antigen DBP-BSA that dibutyl phthalate has been prepared by the identification of uv scan method.
2. the anti-dibutyl phthalate DBP monoclonals of hybridoma cell strain DBP-4E10 secretions described in claim 1 are anti-
Body, it is characterised in that：It is generated secreted by the hybridoma cell strain DBP-4E10.
3. the application of the anti-dibutyl phthalate DBP monoclonal antibodies described in claim 2, it is characterised in that in adjacent benzene two
Application in the DBP analysis detections of formic acid dibutyl ester, has preferable affinity to dibutyl phthalate DBP and detects sensitive
Degree, for dibutyl phthalate DBP immune detections in food security.
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