CN108059620A - Trimethoprim class drug haptens, the monoclonal antibody for detecting trimethoprim class drug and its application - Google Patents
Trimethoprim class drug haptens, the monoclonal antibody for detecting trimethoprim class drug and its application Download PDFInfo
- Publication number
- CN108059620A CN108059620A CN201711140488.8A CN201711140488A CN108059620A CN 108059620 A CN108059620 A CN 108059620A CN 201711140488 A CN201711140488 A CN 201711140488A CN 108059620 A CN108059620 A CN 108059620A
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- China
- Prior art keywords
- trimethoprim
- monoclonal antibody
- class drug
- tmp
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/48—Two nitrogen atoms
- C07D239/49—Two nitrogen atoms with an aralkyl radical, or substituted aralkyl radical, attached in position 5, e.g. trimethoprim
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention discloses a kind of broad-spectrum monoclonal antibodies that can identify a variety of trimethoprim class drugs, it is as secreted by hybridoma cell strain TMP/2G1, the hybridoma cell strain TMP/2G1, is preserved in China typical culture collection center, and preserving number is CCTCC NO:C201783.The invention also discloses the enzyme-linked immunoassay methods and its kit of trimethoprim class medicament residue in detection milk, honey and edible animal tissue.Monoclonal antibody sensitivity prepared by the present invention is higher, and broad spectrum activity is strong, and the accuracy of detection is high, and precision is good, has many advantages, such as easy, quick, easy to operate.
Description
Technical field
The invention belongs to wild animal resources and immunological technique field, and in particular to a kind of to identify trimethoprim class medicine
The broad-spectrum monoclonal antibody of object, the invention further relates to the haptens for being used to prepare the monoclonal antibody and for detecting trimethoprim class medicine
The enzyme-linked immunoassay method (ELISA) and kit of object.
Background technology
Trimethoprim class drug is mainly used as the antibacterial action of enhancing antibiotic, therefore also referred to as Trimethoprim.It is this kind of
It is not general that drug mainly includes trimethoprim (TMP), NSC 408735 (DVD), Ormetoprim (OMP), Baquiloprim (BQP), bromine
Woods (BMP), in addition with the Chinese mugwort for being in research and development state general woods (ADP).Wherein DVD, BQP, OMP are animal specific antibacterial
Medicine is mainly provided commonly for the treatment of various diseases with sulfa drug.Applications of the TMP in animal doctor and people hospital face is very extensive,
The infection of various bacterial diseases can be prevented and treated with other medicines use in conjunction.BMP is on people doctor using relatively broad.This
A little drugs are there are many adverse reaction, such as have negative effect to hemopoietic system.It is many country and organized to set up such drug most
High residue is limited the quantity.Chinese and EU provides maximum residue limit of the TMP in edible animal tissue as 50 μ g/kg, and BQP exists
The maximum residue limit of each target tissue of ox, pig is 10-300 μ g/kg；Canada regulation OMP is in dog salmon edible tissue
Maximum residue limit is 100 μ g/kg；Japan regulation DVD is 50 μ g/kg in the maximum residue limit of edible chicken tissues.
Method for detecting trimethoprim class drug mainly has instrumental method and immunological detection method.Instrumental Analysis
The sensitivity of method, accuracy, precision are higher, and can simultaneously a variety of trimethoprim class drugs be carried out with qualitative, quantitative study, and examined
It surveys limit and has reached μ g/kg or μ g/L levels.But such method needs expensive instrument and equipment, and sample pre-treatments are complex, right
The technology of operating personnel is more demanding, and is only applicable to laboratory research and is not suitable for the screening of high-throughput sample.For examining
Surveying the immunological method of trimethoprim class drug mainly includes colloidal gold immunity chromatography (GICA) and enzyme linked immunosorbent assay
(ELISA).Wherein ELISA method has easy to operate, quick, high specificity, high sensitivity and can carry out half-quantitative detection etc.
Advantage is very suitable for the high-throughput sample detection at scene.
104370829 A of CN disclose a kind of trimethoprim haptens, it is to connect the amino in trimethoprim 2
Upper succinic acid, polyclonal antibody or monoclonal antibody with being used to prepare detection trimethoprim after carrier protein couplet, detection limit
For 0.47ng/mL, the cross reacting rate of each analog is respectively less than 5%.105652004 A of CN disclose another trimethoprim
Haptens, it is to be used to prepare detection methoxy after propionic acid, with carrier protein couplet in connection on 4 methoxyl groups of trimethoprim
The monoclonal antibody of benzyl pyridine, sensitivity is up to 2ppb, with other common equal no cross reactions of sulfa drugs.
The ELISA method of detection trimethoprim class drug can only detect a kind of drugs of TMP at present, almost without on this kind of
The report of more residual ELISA detections of drug.
The content of the invention
The purpose of the present invention is being directed to the defects of existing antibody is only capable of detecting a kind of trimethoprim class drug, a kind of energy is provided
The monoclonal antibody of a variety of trimethoprim class drugs and ELISA method and kit are detected simultaneously, and the present invention also provides a kind of use
In the haptens for preparing monoclonal antibody.
Above-mentioned purpose is achieved through the following technical solutions：
A kind of trimethoprim class drug haptens, shown in structural formula such as formula (1)：
The haptens is that NSC 408735 is substituted by butyric acid by the methyl on 4 by a series of reaction.
The haptens is with, as immunogene, being immunized after animal through cell fusion after carrier protein couplet, obtaining hybridoma
Cell line TMP/2G1, and prepare monoclonal antibody using ascites method is induced in vivo.
The hybridoma cell strain TMP/2G1 is deposited in China typical culture collection center, preserving number CCTCC
NO:C201783。
The preferred bovine serum albumin(BSA) of carrier protein (BSA).
A kind of monoclonal antibody can identify trimethoprim class drug, it is secreted by hybridoma cell strain TMP/2G1.
The monoclonal antibody is used to prepare the kit of detection trimethoprim class drug.
The kit is to detect the enzyme linked immunological kit of trimethoprim class drug.
Application of the kit in the non-diagnostic purpose detection of trimethoprim class drug.
A kind of non-diagnostic purpose enzyme-linked immune detection method of trimethoprim class drug, comprises the following steps：
(1) NSC 408735 is synthesized into haptens shown in formula (1) for raw material by series reaction, then by haptens
Immunogene is obtained after being coupled with bovine serum albumin(BSA)；
(2) coating antigen will be obtained after haptens and chicken ovalbumin coupling；
(3) immunogene obtained with step (1) prepares preserving number as CCTCC NO:The hybridoma cell strain of C201783
TMP/2G1；
(4) monoclonal antibody is prepared with hybridoma cell strain TMP/2G1；
(5) the coating primordial covering solid phase carrier obtained with step (2)；
(6) processing and detection of sample.
The beneficial effects of the invention are as follows：
(1) present invention is when preparing monoclonal antibody, using formula (1) compound represented as haptens, by haptens and
For carrier protein couplet as immunogene, the monoclonal antibody prepared by the immunogene can identify trimethoprim, dimethoxy benzyl simultaneously
Pyridine, end a variety of Trimethoprims of general woods, Baquiloprim, Ormetoprim, brodimoprim；
(2) enzyme-linked immunoassay method of the invention and kit be suitable for detecting milk, honey, edible animal tissue,
The residual of a variety of trimethoprim class drugs, the sensitivity of detection, accuracy are high, and precision is good, trimethoprim, NSC 408735,
End general woods, Baquiloprim, Ormetoprim, the IC of brodimoprim50Value is respectively 0.232,0.527,1.479,4.354,0.965
With 0.119 μ g/L；
(3) detection method according to the present invention is simple, easy to operate, and testing cost is low, operator is required low, and body is good for
Health harm is relatively small.
Description of the drawings
Fig. 1 is the MASS mass spectrograms of haptens.
Fig. 2 is indirect competitive ELISA response curve of the monoclonal antibody with trimethoprim class drug standards of the present invention,
X-axis is trimethoprim concentration of standard solution to numerical value, Y-axis for trimethoprim drug standards solution OD value divided by
" zero " hole OD value (B/B0)。
Specific embodiment
Below by embodiment, the invention will be further described, but does not limit the present invention.
The preparation of 1 immunogene of embodiment and coating antigen
The preparation of 1.1 haptens DVDCOOH
1. weighing 10g DVD, 70mL 48%HBr solution is placed in the round-bottomed flask of 250mL, under the conditions of 100 DEG C of oil baths
Back flow reaction 30min.The sodium hydroxide of 50mL 50% is added in, is stood overnight at room temperature, substantial amounts of white solid is had and is precipitated.
It filters, solid is dissolved in boiling water, and it is neutral to adjust solution with ammonium hydroxide, it is brilliant that white is precipitated in recrystallization under condition of ice bath
Body.2. weighing white crystal 4.5g, 20mL dimethylformamide (DMF), sodium hydrogen 1.5g, 4- bromobutyrate 10g is placed in 50ml
In round-bottomed flask, 12h is reacted at room temperature.400mL water and 20mL n-butanols and 80mL ethyl acetate are added in, is so extracted repeatedly
3 times, collect organic phase.50 DEG C of revolving organic solvents, finally obtain tan solid.With dichloromethane：Methanol=10:1 makees
Pillar purified product is crossed for solvent, finally obtains 2.3g white solids.3. weigh product 2g, 18mL methanol, 10% hydrogen of 2mL
Sodium hydroxide solution is placed in 50mL round-bottomed flasks, and 55 DEG C of reaction 1h, are 4~5 with dilute hydrochloric acid tune solution PH, the meeting under condition of ice bath
It is haptens DVDCOOH to have white crystal to be precipitated.
Through Mass Spectrometric Identification, under positive ion mode bombardment, [M+H]+theoretical value 333.1557, measured value is
(333.1573 see Fig. 1).
The preparation of 1.2 comlete antigen DVDCOOH-DCC-BSA/OVA
The DMF that 10mg DVDCOOH are dissolved in 800 μ L is weighed, then adds in 7mg dicyclohexylcarbodiimides (DCC), 14mg
N-hydroxysuccinimide (NHS), room temperature activation 11h, adds in the BSA 25mg that 7mL PBS dissolve, ice bath under condition of ice bath
Under the conditions of be protected from light 8h.Reaction solution is transferred in bag filter, dialyse 5d in 4 DEG C of PBS, replaces dialyzate three times, dialysis daily
Afterwards, 8000r/min centrifuges 10min, retains supernatant to get DVDCOOH-DCC-BSA, is saved backup in -20 DEG C.Similarly, by BSA
OVA is changed up to coating antigen DVDCOOH-DCC-OVA.
The preparation of 2 monoclonal antibody of embodiment
2.1 animal immune
Using inventor prepare immunogen immune female Balb/C mouse, immunizing dose be respectively every mouse 50,
100th, 4 mouse are immunized in 150 μ g, each dosage group.Immune programme is：Fundamental immunity is complete by immunogene and isometric Freund
After adjuvant emulsion, in the subcutaneous multi-point injection of mouse back, use Freund instead and not exclusively help booster immunization at interval of 2 weeks once later
Agent emulsifies.Most after merging the intraperitoneal injection of first three day, reinforced immunological, amount of antigen doubles, and is not added with adjuvant.
2.2 cell fusions and cloning
During fusion, the Balb/C mouse for last reinforced immunological of learning from else's experience one, eye socket sacrificed by exsanguination (collects serum, is sun
Property serum), in 75% alcohol impregnate 5min disinfection.Sterile taking-up mouse spleen, isolates splenocyte, and freshly prepared
SP2/0 myeloma cell's (SP2/0 myeloma cell comes from this laboratory) presses 1~2 × 107It is a and 108A immune spleen cell,
Ratio is 1:5~1:It between 10, is all placed in 50mL centrifuge tubes, cell is resuspended with the RPMI-1640 basal liquids of 15mL,
1500r min-15min is centrifuged, washes cell 1 time.The gap of centrifugation is put by the culture medium of warm bath, water and 50% polyethylene glycol (PEG)
Enter super-clean bench.The blotting paper of sterilizing is then taken out, after being emptied on the centrifuge tube equipped with myeloma cell and immune spleen cell to the greatest extent,
It tips upside down on and water droplet is drained on blotting paper, tapping tube bottom loosens cell.Timer is opened, draws the PEG of 0.8mL, it is hand-held to be equipped with
The centrifuge tube of cell mixing places it in a moment in water-bath, PEG is slowly added drop-wise on cell mixing, side edged is gently
It stirs, is added in 1min, persistently stir 10s.Then 10mL basic culture solutions are added in, are added slowly to along tube wall on fused cell,
Side edged gently shakes and (cannot blow and beat), and 5mL is added in 3min, 10mL is added in 5min, finally adds basic culture solution
To 40mL, after capping, overturn several times repeatedly, make cell mixing.800r min-15min is centrifuged, abandons supernatant.It draws thin containing raising
The HAT culture medium 5mL of born of the same parents, are slowly added to, and are gently agitated for, and avoid piping and druming；Then the cell is slowly dropped into containing feeder cells
Serum bottle in, after mixing by cell inoculation on Tissue Culture Plate, drip per hole two, be placed in incubator and cultivate.Once
Fusion can be inoculated with 5~7 piece of 96 orifice plate.As needed also can less kind, the general cell number by SP2/0 calculates, per hole inoculum concentration about
Containing 104Left and right SP2/0 cells.In 37 DEG C, 5%CO2It is cultivated in incubator.
It is calculated as 0d on the day of fusion, preceding 3d tries not kinetocyte plate, keeps incubator homeostasis.3d is mended per hole
Add 1 drop HAT complete mediums；5d suctions out l/2 culture supernatants (100 μ L) per hole, adds 1 drop HT complete mediums；After
L/2-3/4 culture supernatants ibid are sucked every 2d, HT complete mediums are changed to after 7d.
Cell colony length to be fused is screened to 1/4 or so of culture hole with the indirect ELISA method of foundation.With zero
Drug hole is compared, and drug hole OD values repressed can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, 2~4 are selected
The cell hole of only 1~2 single colony of a strong positive, cloning is carried out using limited dilution method.
By 3~4 time clonings, the hybridoma cell strain for secreting trimethoprim class anti-drug monoclonal antibody is finally filtered out,
Applicant is named as TMP/2G1, and is sent on May 22nd, 2017 in the Chinese Typical Representative in the Wuhan University of Wuhan City, Hubei Province
Culture collection (CCTCC) preservation, preserving number are CCTCC NO:C201783.By the cell line through Balb/C is injected intraperitoneally
Mouse produces monoclonal antibody.Using the mouse monoclonal antibody Rapid ELISA sizing purchased from Thermo Sxientific companies
Kit identifies that result is mouse IgG to the hypotype and light chain of the obtained monoclonal antibody of the present invention1Hypotype.
The foundation of 3 TMP racing ELISA detecting methods of embodiment
The preparation of 3.1 reagents (reagent that the present embodiment uses is prepared in addition to another indicate using following methods)
Phosphate buffer：NaCl 8.0g, KH2PO40.2g, Na2HPO4.12H2O 2.9g, KCl 0.2g, add tri-distilled water
To 1000mL, pH to 7.4 is adjusted；
Coating buffer：Take Na2CO31.59g NaHCO32.93g adds tri-distilled water to adjust pH value to 9.6 to 1000mL；
Cleaning solution：NaCl 8.0g, KH2PO40.2g, Na2HPO4.12H2O 2.9g, KCl 0.2g, Tween 20
0.5mL adds tri-distilled water to adjust pH to 7.4 to 1000mL；
Confining liquid：Ovalbumin 10.00g is dissolved in 1000mL phosphate buffers；
Substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 160mg, tetrabutyl hydroboration ammonia 21mg are added in
10mL dimethylacetamide amine solvent mixings；
Substrate solution B：Citric acid 13.70g, trisodium citrate 10.14g, carbamide peroxide 282.00mg add tri-distilled water extremely
1000mL；
Substrate cocktail：By A liquid and B liquid by volume 1:100 mix to obtain the final product, now with the current；
Terminate liquid：2mol L-1Sulfuric acid solution.
3.2 coating original contents and antibody working concentration primarily determine that
Coating original content and antibody working concentration are primarily determined that according to square formation titration.Select the DVDCOOH- of above-mentioned synthesis
DCC-OVA is diluted to 800 μ g L as coating antigen with CBS-1、400μg L-1、100μg L-1、50μg L-1、25μg L-1、
12.5μg L-1、6.25μg L-1Then 8 concentration longitudinally add in each concentration in ELISA Plate, each one row of coating put 4 DEG C
Wet box is incubated 12h.Washing 3 times, pats dry, and adds in confining liquid 250 μ L, 37 DEG C of closing 60min.Washing 5 times, pats dry, per Kong Xianjia
Enter the PBS of 50 μ L, then monoclonal antibody dilutes to 1000 respectively with PBS, 2000,4000,8000,16000,32000,
64000th, 128000 times, ELISA Plate is laterally added in, per hole 50 μ L, 37 DEG C of incubation 30min.Washing 5 times, pats dry, HRP is marked sheep
Anti- mouse secondary antibody is diluted to working concentration 1 with PBS：5000, ELISA Plate, 37 DEG C of incubation 30min are added in per 100 μ L of hole.Washing 5 times,
It pats dry；Each hole adds in 100 μ L Substrate cocktails, is protected from light colour developing 15min, 50 μ L terminate liquids is added in, with automatic microplate reader in 450nm
OD value (OD values) is measured at wavelength, selection OD values are close to 2.0, antigen corresponding with the different more significant hole of adjacent holes OD value differences
Concentration and antibody dilution combination are coated with, the results are shown in Table 1.
The result shows that the coating concentration for primarily determining that coating antigen DVDCOOH-DCC-OVA is 100 μ g L-1Or 50 μ g L-1,
Antibody working concentration is 1:16000 or 1:8000.
1 monoclonal antibody square formation of table titrates
3.3 optimal coating original contents and antibody working concentration determine
With the coating concentration for the coating antigen that square formation titration primarily determines that, i.e. 100 μ g L-1、50μg L-12 concentration coatings
ELISA Plate.TMP standard solution is diluted to 0.0125,0.05,0.2,0.8,3.2 μ g L with phosphate buffer-15 it is dense
Degree, 1 is diluted to by monoclonal antibody respectively with phosphate buffer:16000 and 1:8000, adding in zero hole of 50 μ L PBS and 50 μ L
Respectively 50 μ L monoclonal antibodies is added to carry out indirect competitive ELISA measure in the drug hole of TMP standard solution.With TMP concentration of standard solution
1000 times are made abscissa to numerical value, with the ratio (B/B of the OD values in drug hole and " zero " hole OD values0) inhibit as ordinate drafting
Curve selects " 0 " hole OD values close to TMP concentration (IC during 2.0,50% inhibition of generation50) junior's conduct coating concentration.With optimal
Original content coated elisa plate is coated with, TMP standard solution is diluted to 0.0125,0.05,0.2,0.8,3.2 μ g L-15 concentration,
By antibody to select concentration 1:16000 equal difference set dilution factor, are added separately to the TMP standard solution of zero hole and series concentration
Indirect competitive ELISA is carried out in drug hole, draw standard curve and calculates IC50Value." 0 " hole OD values are selected close to 2.0, IC50Compared with
Low person is as optimum antibody working concentration.It the results are shown in Table 2,3.The result shows that most preferably coating concentration is 100 μ g L-1, optimum antibody
Dilution factor is 1:16000.
The most preferably coating concentration of table 2 determines
| It is coated with original content (μ g/L) | Antibody dilution (1：X) | 0 hole OD values | IC50It is worth (μ g/L) |
| 50 | 8000 | 2.52 | 0.517 |
| 100 | 16000 | 2.18 | 0.235 |
3 optimum antibody dilution factor of table determines
The foundation of 3.4 standard curves
By TMP standard solution phosphate buffered saline into 0.0125,0.05,0.2,0.8,3.2 μ g L-15 it is dense
Degree, each concentration repeat 5 holes, are measured according to indirect competitive ELISA method, replication 5 times.With the logarithm of TMP solution concentrations
It is worth for abscissa, B/B0 draws standard curve for ordinate and calculates IC50.It is fitted using ELISA Calc softwares, as a result
As shown in Figure 2.The regression equation of standard curve is y=(A-D)/[1+ (1000x/C)^B]+D, A=0.9244, B=6.5644, C
=2.34793, D=0.09546, r2=1.00000, IC50It is worth for 0.232 ± 0.007 μ g/L (n=5), the range of linearity is
0.0125~3.2 μ g/L.
3.5 cross reactions are tested
By NSC 408735 (DVD), the general woods (ADP) that ends, Baquiloprim (BQP), Ormetoprim (OMP), brodimoprim
(BMP), sulfamethyldiazine (SMR), sulphadiazine (SDZ), sulphathiazole (STZ), sulfaquinoxaline (SQ) and nefrosulfin are rattled away
Piperazine (SCP), into debita spissitudo, the IC of each drug is measured with the ELISA method of foundation with phosphate buffered saline50Value, each medicine
3 multiple holes of object, using monoclonal antibody to the cross reacting rate of trimethoprim as 100%, according to formula 1 calculate monoclonal antibody pair
The cross reacting rate of other 10 categories of drugs, the results are shown in Table 4.The result shows that the antibody can recognize that 6 kinds of trimethoprim class drugs, with
Sulfa drugs then no cross reaction.
4 monoclonal antibody of table is to the cross reacting rate of TMP analogs
| Drug | IC50(μg/L) | Cross reacting rate (%) |
| TMP | 0.232 | 100 |
| DVD | 0.527 | 44.02 |
| ADP | 1.479 | 15.69 |
| BQP | 4.354 | 5.328 |
| OMP | 0.965 | 24.041 |
| BMP | 0.119 | 194.958 |
| SMR | - | - |
| SDZ | - | - |
| STZ | - | - |
| SQ | - | - |
| SCP | - | - |
The assembling of 4 ELISA kit of embodiment
4.1 ELISA kits of the present invention are made of following part：
1) it is coated with the solid phase carrier (ELISA Plate) of coating antigen DVDCOOH-DCC-OVA；
2) 6 bottles of TMP standard solution, concentration are respectively 0,0.0125,0.05,0.2,0.8,3.2 μ g L-1；
3) monoclonal antibody of hybridoma TMP/2G1 secretions；
4) the sheep anti-mouse igg antibody working solution of horseradish peroxidase (HRP) mark；
5) concentrated phosphoric acid salt buffer：NaCl 80.0g, KH2PO42.0g, Na2HPO4.12H2O 29.0g, KCl 2.0g,
Add distilled water to 1000mL；
6) concentrated cleaning solution：NaCl 80.0g, KH2PO42.0g, Na2HPO4.12H2O 29.0g, KCl 2.0g, Tween
20 5mL add tri-distilled water to 1000mL；
7) substrate solution A：3,3', 5', 5- tetramethyl biphenyl diamines (TMB) 160mg, tetrabutyl hydroboration ammonia 21mg are added in
10mL dimethylacetamide amine solvent mixings；
8) substrate solution B：Citric acid 13.70g, trisodium citrate 10.14g, carbamide peroxide 282.00mg add tri-distilled water extremely
1000mL；
9) terminate liquid：2mol L-1Sulfuric acid solution.
The preparation of 4.2 ELISA Plates
DVDCOOH-DCC-OVA is diluted to 100 μ g L with coating buffer-1, 100 μ L are added in per hole, 37 DEG C are incubated 12h, incline
Coating buffer is gone, 250 μ L cleaning solutions are added in per hole and are washed 3 times, are patted dry, 250 μ L of confining liquid, 37 DEG C of incubations are then added in per hole
120min, liquid in hole of inclining, cleaning solution are washed 3 times, patted dry, and are preserved with masking foil vacuum sealing.
The mensuration program of 5 enzyme linked immunological kit of embodiment
5.1 the preparation of reagent
1) sample diluting liquid：It is used after the concentrated phosphoric acid salt buffer provided in kit is diluted 10 times with tri-distilled water.
2) cleaning solution：It is used after the cleaning solution provided in kit is diluted 10 times with tri-distilled water.
3) Substrate cocktail：According to each institute's expense, the substrate solution A of preparation and substrate solution B is pressed into volume 1:100 is mixed
It is even, it is now with the current.
5.2 sample pre-treatments
Milk：6mL Fresh Milks is taken to centrifuge 10min through 6000r/min under the conditions of 50mL centrifuge tubes, 4 DEG C, suck upper strata
Fat takes interlayer emulsion PBST to be measured after diluting 10 times for ELISA.
Honey：Honey 2.0g is weighed, the drug that 0.02 μ L have diluted is added in, is fully vortexed, after concussion, dilute 10 with PBST
It is measured after times for ELISA.
Edible tissue：Weigh homogeneous good fresh edible tissue (chicken, chicken gizzard, pork, pork liver, Ren sus domestica)
2.0g adds in the drug that 20 μ L have diluted, is fully vortexed, after concussion, adds in 8mL acetonitriles, 8000r/mim is centrifuged after being fully vortexed
15min.Take supernatant N2Drying is redissolved with 2mL containing 10% methanol PBST, and then ELISA is measured.
5.3 determination step
1) it is loaded：50 μ L of trimethoprim series concentration standard solution or sample extracting solution are added in into ELISA Plate micropore, so
50 μ L of monoclonal antibody working solution are added in afterwards, are placed in wet box, 37 DEG C of constant-temperature incubation 30min；
2) wash：The liquid in hole is poured out, adding in 250 μ L of cleaning solution in every hole washs 3 times and pat dry；
3) the sheep anti-mouse igg antibody working solution of horseradish peroxidase (HRP) mark is added：Horseradish peroxidating is added in per hole
The 100 μ L of sheep anti-mouse igg antibody working solution of object enzyme (HRP) mark, are placed in wet box, 37 DEG C of constant-temperature incubation 30min；
4) wash：The liquid in hole is poured out, 250 μ L of cleaning solution is added in every hole, washs 3 times and pat dry；
5) substrate is added：100 μ L of Substrate cocktail are added in per hole, are placed in wet box, 37 DEG C of constant-temperature incubation 15min；
6) terminate liquid is added：50 μ L of terminate liquid are added in per hole；
7) measure：The OD value (OD values) in every hole is measured at 450nm with microplate reader.
5.4 results judge
Standard curve：
With the standard items OD values divided by " zero " hole OD values (B/B measured0) it is ordinate, trimethoprim (TMP) concentration
Standard curve is made for abscissa to numerical value, and carries out linear regression, draws regression equation.
The concentration of TMP calculates in milk, honey, edible animal tissue and milk：
The inhibiting rate (the OD values of the sample obtained divided by " zero " hole OD values) of sample is calculated, substitutes into the recurrence of standard curve
Equation calculation TMP concentration.
Other trimethoprim class drug concentrations calculate in milk, honey, edible animal tissue：
The inhibiting rate (the OD values of the sample obtained divided by " zero " hole OD values) of sample is calculated, substitutes into the recurrence of standard curve
In equation, the concentration of other trimethoprim class drugs is converted to according to formula 2.
The sensitivity of 6 ELISA method of the present invention of embodiment, precision, accuracy test
The sensitivity test of 6.1 kits of the present invention
By 20 part of 0 μ g L-1The detection OD values of standard solution substitute into standard curve, calculate 20 parts of blank determination concentration, are obtained
Average value and standard deviation calculate Z values according to formula Z=C+3SD, and the sensitivity of the ELISA method is 0.053 μ g L-1.Minimum inspection
Limit (LOD) is surveyed to determine by following steps：The OD values of 20 parts of blank tissue samples are measured, according to the regression equation meter of standard curve
Corresponding TMP concentration is calculated, then calculates the average value of TMP concentrationWith standard deviation (SD), according to formulaCalculate the LOD in sample；Quantitative limit6 kinds of trimethoprim are in different samples
LOD and LOQ refer to table 5.
Detection limit and quantitative limit of the 5 trimethoprim class drug of table in each sample
The precision test of 6.2 kits of the present invention
TMP standard items are diluted to 0,0.0125,0.5,0.2,0.8,3.2 μ g L-16 concentration, per 5 repetitions of concentration,
According to indirect competitive ELISA method replication 5 times, it is molten to go out each concentration TMP standards using the regression equation calculation of standard curve
The measured value of liquid, the interior coefficient of variation between plate of computing board, the results are shown in Table 6.Within-run and between-run analysis coefficient is respectively less than 10%, precision
Degree is higher.
The interior coefficient of variation between plate of the plate of 6 standard curve of table
The accuracy of 6.3 kits of the present invention, repetitive test
6 kinds of trimethoprim class drug standards of three concentration are added in 6 kinds of samples by this research respectively, as a result such as table
Shown in 7-12.In the range of 86.3%~107.7%, the coefficient of variation is less than TIANZHU XINGNAO Capsul of such drug in milk
13.0%, the TIANZHU XINGNAO Capsul in honey is in the range of 87.4%~104.7%, and the coefficient of variation is less than 16.4%, in chicken
In TIANZHU XINGNAO Capsul in the range of 82.2%~97.7%, the coefficient of variation is less than 12.6%, addition in chicken liver recycling
For rate in the range of 83.2%~97.7%, the coefficient of variation is less than 12.2%, TIANZHU XINGNAO Capsul in pork 83.0%~
In the range of 96.3%, the coefficient of variation is less than 19%, and the TIANZHU XINGNAO Capsul in pig liver becomes in the range of 81.4%~96.8%
Different coefficient is less than 15.5%.According to the requirement of the Ministry of Agriculture of China, the rate of recovery scope of residue of veterinary drug enzyme linked immunological kit exists
Between 60%~120%, batch in and batch between the coefficient of variation be below or equal to 25% and 30%.
TIANZHU XINGNAO Capsul of the 7 trimethoprim drug of table in milk sample
TIANZHU XINGNAO Capsul of the 8 trimethoprim drug of table in honey sample
TIANZHU XINGNAO Capsul of the 9 trimethoprim drug of table in chicken meat sample
TIANZHU XINGNAO Capsul of the 10 trimethoprim drug of table in chicken liver sample
TIANZHU XINGNAO Capsul of the 11 trimethoprim drug of table in pork sample
TIANZHU XINGNAO Capsul of the 12 trimethoprim drug of table in pig liver sample
Claims (9)
1. a kind of trimethoprim class drug haptens, shown in structural formula such as formula (1)：
2. application of the haptens described in claim 1 in the monoclonal antibody for preparing detection trimethoprim class drug.
3. a kind of monoclonal antibody for detecting trimethoprim class drug, it is characterised in that：It is anti-by described in claim 1 half
It is former with as immunogene, be immunized after carrier protein couplet after animal through cell fusion, obtain hybridoma cell strain TMP/2G1, and
It is prepared using ascites method is induced in vivo.
4. the hybridoma cell strain TMP/2G1 described in claim 3, is deposited in China typical culture collection center, preservation
Number be CCTCC NO:C201783.
5. application of the monoclonal antibody in the kit for preparing detection trimethoprim class drug described in claim 3.
6. including the kit of monoclonal antibody described in claim 3, which is the enzyme-linked of detection trimethoprim class drug
Immune reagent kit.
7. application of the kit in the non-diagnostic purpose detection of trimethoprim class drug described in claim 6.
8. a kind of non-diagnostic purpose enzyme-linked immune detection method of trimethoprim class drug, it is characterised in that comprise the following steps：
(1) haptens described in claim 1 and chicken ovalbumin are coupled to obtain coating antigen；
(2) it is CCTCC NO with preserving number:C201783 hybridoma cell strains TMP/2G1 prepares monoclonal antibody；
(3) the coating primordial covering solid phase carrier of step (1) is used；
(4) processing and detection of sample.
9. according to the method described in claim 8, it is characterized in that：The sample includes milk, honey and edible animal group
It knits.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112939873A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof |
| CN112939875A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPO, artificial antigen, antibody and preparation method and application thereof |
| CN113281517A (en) * | 2021-05-10 | 2021-08-20 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immunomagnetic bead pretreatment with HPLC-UV |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104341357A (en) * | 2014-09-29 | 2015-02-11 | 江南大学 | Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen |
| CN104370829A (en) * | 2014-09-29 | 2015-02-25 | 江南大学 | Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen |
| CN105652004A (en) * | 2015-12-29 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten |
-
2017
- 2017-11-16 CN CN201711140488.8A patent/CN108059620A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104341357A (en) * | 2014-09-29 | 2015-02-11 | 江南大学 | Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen |
| CN104370829A (en) * | 2014-09-29 | 2015-02-25 | 江南大学 | Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen |
| CN105652004A (en) * | 2015-12-29 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Trimethoprim hapten as well as colloidal gold detection device and preparation method of trimethoprim hapten |
Non-Patent Citations (2)
| Title |
|---|
| DRIES J. H. DE CLERCQ，ET AL.: "Alternative Reagents for Methotrexate as Immobilizing Anchor Moieties in the Optimization of MASPIT: Synthesis and Biological Evaluation", 《CHEMBIOCHEM》 * |
| 英瑜: "水产品中磺胺二甲嘧啶间接竞争ELISA（ciELISA）检测法的建立及初步应用", 《万方学位论文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112939873A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof |
| CN112939875A (en) * | 2021-02-08 | 2021-06-11 | 华南农业大学 | Trimethoprim hapten TMPO, artificial antigen, antibody and preparation method and application thereof |
| CN112939873B (en) * | 2021-02-08 | 2022-03-25 | 华南农业大学 | Trimethoprim hapten TMPH, artificial antigen, antibody and preparation method and application thereof |
| CN112939875B (en) * | 2021-02-08 | 2022-05-20 | 华南农业大学 | Trimethoprim hapten TMPO, artificial antigen, antibody and preparation method and application thereof |
| CN113281517A (en) * | 2021-05-10 | 2021-08-20 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immunomagnetic bead pretreatment with HPLC-UV |
| CN113281517B (en) * | 2021-05-10 | 2024-01-30 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immune magnetic bead pretreatment with HPLC-UV |
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