CN118084965A - Long-acting phosphorus hapten, antigen, antibody, detection device and preparation and application thereof - Google Patents
Long-acting phosphorus hapten, antigen, antibody, detection device and preparation and application thereof Download PDFInfo
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- CN118084965A CN118084965A CN202410502045.2A CN202410502045A CN118084965A CN 118084965 A CN118084965 A CN 118084965A CN 202410502045 A CN202410502045 A CN 202410502045A CN 118084965 A CN118084965 A CN 118084965A
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 78
- 239000011574 phosphorus Substances 0.000 title claims abstract description 78
- 238000001514 detection method Methods 0.000 title claims abstract description 52
- 239000000427 antigen Substances 0.000 title claims abstract description 36
- 102000036639 antigens Human genes 0.000 title claims abstract description 36
- 108091007433 antigens Proteins 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title abstract description 17
- KRTSDMXIXPKRQR-AATRIKPKSA-N monocrotophos Chemical compound CNC(=O)\C=C(/C)OP(=O)(OC)OC KRTSDMXIXPKRQR-AATRIKPKSA-N 0.000 claims abstract description 50
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 27
- 230000002085 persistent effect Effects 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 17
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 8
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 8
- 241001465754 Metazoa Species 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 238000000576 coating method Methods 0.000 claims description 27
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- 238000004587 chromatography analysis Methods 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 10
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- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 7
- JRNVZBWKYDBUCA-UHFFFAOYSA-N N-chlorosuccinimide Chemical compound ClN1C(=O)CCC1=O JRNVZBWKYDBUCA-UHFFFAOYSA-N 0.000 claims description 6
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 6
- 230000001900 immune effect Effects 0.000 claims description 6
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- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 claims description 3
- HAAUVXXFRQXTTQ-UHFFFAOYSA-N 2-[4-(azaniumylmethyl)phenyl]acetate Chemical compound NCC1=CC=C(CC(O)=O)C=C1 HAAUVXXFRQXTTQ-UHFFFAOYSA-N 0.000 claims description 3
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims description 3
- JKUYRAMKJLMYLO-UHFFFAOYSA-N tert-butyl 3-oxobutanoate Chemical compound CC(=O)CC(=O)OC(C)(C)C JKUYRAMKJLMYLO-UHFFFAOYSA-N 0.000 claims description 3
- CYTQBVOFDCPGCX-UHFFFAOYSA-N trimethyl phosphite Chemical compound COP(OC)OC CYTQBVOFDCPGCX-UHFFFAOYSA-N 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
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- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- -1 MCP-H-2 Chemical compound 0.000 description 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- NNKVPIKMPCQWCG-UHFFFAOYSA-N methamidophos Chemical compound COP(N)(=O)SC NNKVPIKMPCQWCG-UHFFFAOYSA-N 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a long-acting phosphorus hapten, an antigen, an antibody, a detection device and preparation and application thereof, and relates to a long-acting phosphorus hapten, an antigen, an antibody, a colloidal gold detection device and methods for preparing and detecting long-acting phosphorus in agricultural products. The invention provides a method for preparing artificial antigen by hapten synthesis and antigen coupling carrier protein of monocrotophos, and an antibody which is specifically aimed at the monocrotophos and is produced by a body of an animal after the animal is immunized, wherein the IC 50 value is 7.59 ng/mL, the linear detection range (IC 20-IC80) of the monocrotophos is 2.28-25.23ng/mL, and the method accords with the limit regulation of the monocrotophos in national standard GB 2763-2021 maximum pesticide residue limit in food safety national standard food. Therefore, the invention can be applied to the rapid detection of the residual content of the persistent phosphorus in the agricultural products.
Description
Technical Field
The invention relates to the technical field of agricultural product safety detection, in particular to a long-acting phosphorus hapten, an antigen, an antibody, a detection device and preparation and application thereof.
Background
The immunological detection analysis method organically combines chemical synthesis, biotechnology and physicochemical test means, and has the characteristics of strong specificity, high sensitivity, convenience, high analysis capacity, low analysis cost, low requirement on the degree of instrumentation and the like. Has wide application prospect and development potential in the aspects of analysis and on-site rapid detection of ultra-trace pesticide residues in complex samples. In recent years, the immunity detection technology has been increasingly researched in the aspect of pesticide residue analysis, but the immunity detection method for the persistent phosphorus is not seen at home and abroad, only glutaraldehyde is used as a cross-linking agent to prepare the persistent phosphorus artificial antigen and the coating antigen, and the effect of obtaining antibodies through immunization is general. The invention designs and develops a proper phosphorus hapten with high sensitivity by adopting a novel synthesis method, prepares a phosphorus antibody with high sensitivity, establishes a corresponding rapid detection method for phosphorus, and realizes rapid detection of phosphorus residue in agricultural products by an immunological method.
Disclosure of Invention
The invention aims to provide a phosphorus hapten, an antigen, an antibody, a detection device, and preparation and application thereof.
According to one aspect of the invention, there is provided an immune hapten of monocrotophos having the structure shown in formula (I):
Formula (I).
According to another aspect of the present invention, there is provided a coating hapten of monocrotophos having a structure as shown in formula (II):
formula (II).
According to a further aspect of the present invention there is provided a method of preparing an immune hapten of penstock or a coated hapten of penstock comprising the steps of:
S1, performing halogen substitution reaction on tert-butyl acetoacetate and N-chlorosuccinimide to obtain an intermediate 1, wherein the structural formula of the intermediate 1 is shown in a reaction formula (III):
Formula (III);
s2, carrying out Perkow rearrangement reaction on the intermediate 1 and trimethyl phosphite under the condition of heating in dichloroethane to obtain an intermediate 2, wherein the structural formula of the intermediate 2 is shown in a reaction formula (IV):
formula (IV);
S3, reacting the intermediate 2 with trifluoroacetic acid to remove tert-butyl, and obtaining an intermediate 3, wherein the structural formula of the intermediate 3 is shown in a reaction formula (V):
formula (V);
s4, reacting the intermediate 3 with N-hydroxysuccinimide to prepare first active ester, and reacting the first active ester with 4-aminobutyric acid under alkaline conditions to obtain the immune hapten MCP-H-1 of the monocrotophos, wherein the reaction formula is shown in the following formula (VI):
Formula (VI);
s5, reacting the intermediate 3 with N-hydroxysuccinimide under the action of a condensing agent to prepare second active ester, reacting the second active ester with 4-aminomethylphenylacetic acid under an alkaline condition to obtain the coating hapten MCP-H-2 of the persistent phosphorus, wherein the reaction formula is shown in the following formula (VII):
formula (VII).
According to a fourth aspect of the present invention there is provided a phosphorus-long-acting antigen comprising a phosphorus-long-acting immune antigen and a phosphorus-long-acting coated antigen, the phosphorus-long-acting immune antigen being a conjugate of a phosphorus-long-acting immune hapten and a carrier protein, the phosphorus-long-acting coated antigen being a conjugate of a phosphorus-long-acting coated hapten and a carrier protein, the carrier protein being bovine serum albumin, human serum albumin, chicken egg serum albumin or hemocyanin.
According to a fifth aspect of the present invention there is provided the use of an immune hapten of phosphorus sedge or a coated hapten of phosphorus sedge or a phosphorus sedge antigen for non-disease diagnosis in immunological detection of phosphorus sedge.
According to a sixth aspect of the present invention, there is provided a phosphorus-long antibody, which is prepared from a phosphorus-long immune antigen by animal immunization, and which is a phosphorus-long monoclonal antibody.
According to a seventh aspect of the present invention there is provided the use of a phosphorus-long antibody for the diagnosis of a non-disease in the immunological detection of phosphorus-long.
According to an eighth aspect of the invention, there is provided a device for detecting a long-acting phosphorus by colloidal gold chromatography, comprising a test strip and a reaction cup, wherein the test strip comprises a reaction membrane, the reaction membrane is provided with a detection area and a quality control area, the detection area is coated with a long-acting phosphorus coating antigen, and the reaction cup contains a colloidal gold-labeled long-acting phosphorus antibody.
According to a ninth aspect of the present invention, there is provided a method for detecting monocrotophos in a sample, wherein the method comprises detecting monocrotophos in the sample by using a monocrotophos colloidal gold chromatography detection device, and the sample is an agricultural product.
The invention has the beneficial effects that: the preparation method of the monocrotophos hapten provided by the invention has the advantages of easiness in obtaining the used chemical reagent, simple operation process, concise and effective synthesis steps, higher reaction yield and lower detection cost. The invention provides an artificial antigen prepared from immune hapten coupling carrier protein of the persistent phosphorus, and an antibody which is produced by a body of the animal after the animal is immunized and is specific to the persistent phosphorus, wherein the antibody has high titer, an ELISA standard curve is established for the persistent phosphorus antibody, the IC 50 value of the antibody is 7.59 ng/mL, the linear detection range (IC 20-IC80) of the persistent phosphorus is 2.28-25.23ng/mL, and the limit regulation of the persistent phosphorus is met in the national standard GB 2763-2021 (maximum pesticide residue limit in food safety national standard food) issued recently. Therefore, the invention can be applied to the rapid detection of the residual content of the persistent phosphorus in the agricultural products.
Drawings
FIG. 1 is a mass spectrum of an immune hapten of monocrotophos (MCP-H-1) according to one embodiment of the invention.
FIG. 2 is a mass spectrum of a coating hapten of monocrotophos (MCP-H-2) according to one embodiment of the invention.
Figure 3 is an ELISA standard curve based on a juxifos antibody of one embodiment of the invention.
Detailed Description
The application will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the application, as various equivalent modifications of the application will occur to those skilled in the art upon reading the application, as defined in the appended claims. Unless otherwise specified, all materials and reagents of the application are those commercially available in the conventional market.
EXAMPLE 1 Synthesis and identification of immune hapten and coating hapten of monocrotophos
The preparation method of the immune hapten and the coating hapten of the monocrotophos comprises the following steps:
S1, weighing 4.0g of tert-butyl acetoacetate, dissolving with 60mL of chloroform, adding 5.0g of N-chlorosuccinimide in batches at room temperature, reacting for 4.5 hours at room temperature after the addition, evaporating the solvent under reduced pressure after the reaction is finished, and directly purifying by a column to obtain 6.2g of intermediate 1 which is colorless oily substance;
S2, dissolving 6.0g of the intermediate 1 with 50mL of dichloroethane, dripping 3.9g of trimethyl phosphite, slowly heating to 80 ℃ after dripping, reacting for 2 hours, decompressing and evaporating the solvent after the reaction, directly purifying by a column to obtain 5.5g of the intermediate 2, and obtaining colorless oily matter;
S3, adding 5.2g of intermediate 2 into a 100mL single-port bottle, dissolving with 30mL of dichloromethane, adding 15mL of TFA under the condition of stirring at room temperature, reacting for 2 hours at room temperature, distilling under reduced pressure to evaporate the solvent after the raw materials are basically reacted completely, and directly purifying by a column to obtain 4.5g of intermediate 3;
S4, weighing 2.1g of intermediate 3, 1.3g of N-hydroxysuccinimide (NHS) and 2.5g of EDC, dissolving in 30ml of dichloromethane, stirring and reacting overnight to prepare first active ester, then adding 2.0g of triethylamine and 1.5g of 4-aminobutyric acid, continuously reacting for 12 hours, evaporating the solvent, and purifying by a column to obtain the immune hapten of the monocrotophos, namely MCP-H-1;
S5, 2.1g of intermediate 3, 1.3g of N-hydroxysuccinimide (NHS) and 2.5g of EDC.HCl are weighed and dissolved in 30ml of dichloromethane, the mixture is stirred and reacted overnight to prepare second active ester, then 2.0g of triethylamine and 1.8g of 4-aminomethylphenylacetic acid are added for continuous reaction for 12 hours, the solvent is evaporated, and the coating hapten of the monocrotophos, namely MCP-H-2, is obtained through column purification.
The mass spectrometry is adopted to identify the immune hapten of the monocrotophos, and the obtained mass spectrum is shown in figure 1 of the specification. From the mass spectrum, the molecular ion peak of the immune hapten of the monocrotophos is EI-MS (negative) m/z:295.5[ M-H ] - is the target molecular ion peak, which is consistent with the molecular weight 295.08 of the immune hapten of the monocrotophos, which shows that the immune hapten of the monocrotophos shown in the formula (I) is successfully synthesized.
And identifying the coating hapten of the monocrotophos by adopting a mass spectrometry method, wherein the obtained mass spectrum is shown in figure 2 of the specification. From the mass spectrum, the molecular ion peak of the coating hapten of the monocrotophos is EI-MS (negative) m/z:356.99[ M-H ] - is the target molecular ion peak, which is consistent with the molecular weight 357.30 of the coating hapten of the monocrotophos, which shows that the coating hapten of the monocrotophos shown in the formula (II) is successfully synthesized.
The immune hapten and the coating hapten of the persistent phosphorus prepared by the invention introduce a connecting arm structure and active groups for coupling macromolecules on the basis of retaining the basic structure of the persistent phosphorus, so that the coupling of the immune hapten and the active groups of macromolecules is facilitated, the basic structure of the persistent phosphorus with smaller molecular structure and molecular weight can be fully exposed after the coupling, and the influence on the identification of animal organisms caused by the masking of the immune hapten and the coating hapten by the macromolecules is avoided.
EXAMPLE 2 Synthesis of a phosphorus-extended antigen
The preparation method of the persistent phosphorus immune antigen comprises the following steps:
Weighing 45mg of the immune hapten MCP-H-1 of the monocrotophos, dissolving in 2.5ml of LDMF, adding 25mg of NHS and 30mg of EDC.HCl, and reacting for 6 hours at room temperature to prepare an activation solution; 45mg BSA is dissolved in 3mL boric acid buffer solution with the concentration of 0.1M PH9.0, 1mL LDMF and 0.6mL activating solution are added, PBS (0.01 mol/L phosphate buffer solution with the concentration of pH=7.4) is used for dialysis after reaction for 4 hours at room temperature, each 4: 4H is changed for 1 time, the solution is changed for 7-8 times, the solution is centrifuged for 5 minutes at 4000 revolutions per minute after dialysis, and supernatant is taken to prepare the long-acting phosphorus immune antigen, namely the MCP-H-1-BSA conjugate and is stored at the temperature of minus 20 ℃.
And similarly, preparing a coating antigen of the monocrotophos, replacing the immune hapten MCP-H-1 of the monocrotophos with the coating hapten MCP-H-2 of the monocrotophos, replacing BSA with OVA, preparing the coating antigen of the monocrotophos, namely a MCP-H-2-OVA conjugate, and storing at the temperature of minus 20 ℃.
EXAMPLE 3 preparation and purification of monoclonal antibodies to monocrotophos
3.1 Immunization of animals
Healthy BALB/c mice of 6 to 8 weeks of age were selected for immunization, and after the antigen for immunization of phosphorus obtained in example 2 was mixed and emulsified with an equivalent amount of Freund's adjuvant, BALB/c mice were subjected to subcutaneous multipoint injection of the back of the neck (except for sprint immunization). The first immunization is carried out by using complete Freund's adjuvant, and the dosage is 180 mug/dose; boosting is carried out after 4 weeks at a dose of 90 mug/dose, the mixture is mixed and emulsified by incomplete Freund's adjuvant, and then the boosting is carried out for a plurality of times for 3 weeks; the dose is halved again during the sprint immunization, 45 mug/mouse, and the complete antigen is diluted by normal saline for intraperitoneal injection. The tail breaking blood sampling detection can be carried out after the third immunization of the mice, the titer of the serum of the mice and the IC 50 are detected by an indirect competition enzyme-linked immunosorbent assay (IC-ELISA), and the mice with high titer and low IC 50 are selected for fusion;
3.2 Cell fusion and cloning
Spleen cells of immunized BALB/c mice were taken at a ratio of 10:1 are fused with SP2/0 myeloma cells, and a monoclonal hybridoma cell strain of the monoclone phosphorus which stably secretes the monoclonal antibody of the monoclone phosphorus is obtained by screening;
3.3 Cell cryopreservation and resuscitation
The monoclonal hybridoma cells of the monocrotophos are prepared into cell suspension of 5X 10 6/mL by using a frozen stock solution, and the cell suspension is preserved for a long time in liquid nitrogen. Taking out the freezing tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen solution, and transferring into a culture flask for culture;
3.4 Preparation, purification and Performance determination of monoclonal antibodies
Incremental culture method: placing the monoclone phosphorus hybridoma cells in a cell culture medium, culturing at 37 ℃, purifying the obtained culture solution by an octanoic acid-saturated ammonium sulfate method to obtain the monoclone phosphorus antibody, and preserving at-20 ℃. The properties of the antibodies were determined by an indirect ELISA method. ELISA standard curve is established for the antibody of the monocrotophos, and the IC 50 value is 7.59 ng/mL, and the linear range (IC 20-IC80) of the monocrotophos is 2.28-25.23ng/mL, as shown in figure 3 of the specification.
Example 4 preparation of a device for detecting a prolonged phosphorus colloidal gold chromatography
4.1 Preparation of colloidal gold solution
Diluting 1% chloroauric acid solution into 0.01% (mass fraction) by using double distilled deionized water, placing 100mL of 0.01% chloroauric acid solution into a conical flask, heating to boil by using a constant-temperature electromagnetic stirrer, adding 2.0 mL of 1% trisodium citrate solution under continuous high temperature and continuous stirring, continuing to stir and heat at a constant speed until the solution is transparent red, stopping cooling to room temperature, recovering to original volume by using deionized water, obtaining colloidal gold solution, and preserving at 4 ℃. The prepared colloidal gold solution has pure appearance, is transparent and has no sediment or floaters;
4.2 Preparation of monoclonal antibody-colloidal gold marker of monocrotophos
Under the magnetic stirring, regulating the pH value of the colloidal gold solution to 7.2 by using 0.2mol/L potassium carbonate, adding 20-60 mug of the monoclone phosphorus antibody per milliliter of the colloidal gold solution, adding the monoclone phosphorus antibody prepared in the example 3 into the colloidal gold solution, continuously stirring and uniformly mixing for 30min, standing for 10min, adding 10% Bovine Serum Albumin (BSA) solution, and standing for 10min, wherein the volume percentage of the solution in the colloidal gold solution is 1%. Centrifuging at 12000rpm at 4deg.C for 40min, discarding supernatant, and re-suspending the precipitate with 1/10 of the original colloidal gold solution volume of re-dissolving buffer solution to obtain monoclonal antibody-colloidal gold marker, and storing at 4deg.C;
reconstitution buffer: 0.02mol/L phosphate buffer solution containing 0.3-0.5% of bovine serum albumin, 0.1-0.3% of tween-20, 3-6% of trehalose and pH=7.2;
4.3 preparation of microporous reaction cup
Adding 100 mu L of the monoclonal antibody of the phosphorus-colloidal gold into a micropore reaction cup, putting into a freeze dryer, pre-freezing for 3 hours at the temperature of a cold trap of-50 ℃, and then drying for 6 hours in vacuum, thus obtaining the micropore reaction cup of the freeze-dried monoclonal antibody of the phosphorus-colloidal gold, and sealing and preserving, wherein the freeze-drying amount of the monoclonal antibody of the phosphorus-colloidal gold is 0.20-0.50 mu g/ml;
4.4 Preparation of sample absorbent pad
The sample absorption pad is placed in phosphate buffer solution containing 0.02mol/L of bovine serum albumin to be soaked for 2 hours, and is dried for 2 hours at 50 ℃ for standby. The pH of the phosphate buffer solution of 0.02mol/L is 7.2, wherein the volume percentage of the bovine serum albumin is 1.0%;
4.5 Preparation of reaction film
The coating process comprises the following steps: diluting the long-acting phosphorus coated antigen (MCP-H-2-OVA) prepared in example 2 to a concentration of 10mg/ml by using a phosphate buffer solution, and coating the long-acting phosphorus coated antigen on a detection area (T area) of a nitrocellulose membrane by using a gold-labeled gold spot plating instrument to obtain a coating concentration of 0.5 mg/ml; the concentration of the goat anti-mouse antibody (commercial product) was diluted to 10mg/ml with a phosphate buffer solution having a concentration of 0.01 mol/L and a ph=7.4, and coated on a quality control region (region C) on a nitrocellulose membrane with a gold-labeled gold-dot membrane meter at a coating concentration of 1.0 mg/ml. The coated reaction film was dried at 50℃for 6 hours.
4.6 Preparation of a device for detecting long-acting phosphorus by colloidal gold chromatography
4.6.1 Assembly of test strips
Sequentially adhering a sample absorption pad, a reaction membrane and a water absorption pad on a bottom plate, wherein the bottom plate is a PVC bottom plate, the sample absorption pad is a piece of filter paper, the water absorption pad is a piece of filter paper, and the reaction membrane is a nitrocellulose membrane. The end of the sample absorbing pad is connected with the initial end of the reaction membrane, the end of the reaction membrane is connected with the initial end of the water absorbing pad, the initial end of the sample absorbing pad is aligned with the initial end of the bottom plate, and the end of the water absorbing pad is aligned with the end of the bottom plate.
4.6.2 Assembly of test paper box for chromatographic detection of long-acting phosphorus colloidal gold
And (3) assembling the test strip obtained in the step 4.6.1 and the microporous reaction cup obtained in the step 4.3 into a test paper box, storing in an environment of 2-8 ℃ and storing for 12 months in the effective period.
Example 5A method for detecting monocrotophos in a sample
5.1 Sample pretreatment
Weighing 2.0 g+/-0.01 g of homogenized vegetable sample or fruit sample into a 50mL polystyrene centrifuge tube, adding 8mL of sample extracting solution (the sample extracting solution is prepared by uniformly mixing 7mL 0.1M pH 8.5 PBS volumes and 1mL of ethanol), uniformly mixing, centrifuging at room temperature (20-25 ℃) for 1min at 3000r/min, and taking supernatant as a solution to be detected;
5.2 Measurement procedure
200 Μl of the above-mentioned liquid to be tested is sucked into the microporous reaction cup of the chromatographic detection device for the colloidal gold of phosphorus for long-acting prepared in example 4, and sucked up and down for 5-10 times to mix uniformly. Incubating at room temperature for 3min minutes, inserting the test strip into a reaction cup, incubating at room temperature for 3 minutes, taking out the test strip, gently scraping a sample pad at the lower end of the test strip, and judging and reading the result;
5.3 Result determination
The result determination is performed by comparing the color shades of the control line (C line) and the detection line (T line).
Positive: when the quality control area (C) shows a strip, the detection area (T) does not develop color, and the detection area (T) is judged to be positive, namely, the sample has the long-acting phosphorus, and the long-acting phosphorus is indicated by "+";
negative: when the quality control area (C) and the detection area (T) both show strips, judging as negative, namely, the sample does not contain the persistent phosphorus, and the sign is "-;
Invalidation: and when the quality control area (C) does not display a strip, judging that the test paper fails.
Example 6 sensitivity and false negative Rate of a device for detecting a prolonged phosphorus colloidal gold chromatography
Cucumber, green beans and citrus which are tested to contain no persistent phosphorus are selected as blank samples, the highest residual limit (MRL) of the cucumber, the green beans and the citrus is 0.03mg/kg according to the specification in GB 2763-2021, and the detection limit of the method for setting the cucumber, the green beans and the citrus is 0.03mg/kg, namely the concentration of interest. The addition levels were 1-fold concentration of interest and 2-fold concentration of interest, respectively, and the sensitivity and false negative rate were examined. Two samples of additive concentration levels, 50 samples each, were tested as in example 5 and the results are shown in Table 1 below.
TABLE 1 sensitivity and false negative Rate detection results of a detection device for detecting persistent phosphorus by colloidal gold chromatography
As shown in Table 1, the detection sensitivity of the detection method in this example to the residual phosphorus in the sample is not less than 95%, and the false negative rate is not more than 5%.
EXAMPLE 7 false positives and specificity of a device for detecting phosphorus by colloidal gold chromatography
Blank samples of cucumber, green bean and citrus are selected, and a blank matrix labeling mode is adopted to prepare 50 parts of samples with 2 concentration levels (0.5 times of detection limit and blank matrix). Samples were tested using the test method of example 5, the test results are shown in Table 2 below.
TABLE 2 false positive detection results of a long-acting phosphorus colloidal gold chromatography detection apparatus
Selecting long-acting phosphorus structural analogues, such as: the specificity of the test strip is verified by adding standard substances to blank samples of cucumber, green bean and orange, wherein the standard substances have 1-time detection limit (0.03 mg/kg) and 10-time detection limit (0.3 mg/kg), 10 parts of each sample are adopted, and the detection results are shown in the table 3 below.
TABLE 3 specificity detection results of the chromatographic detection device for phosphorus colloid gold
As is clear from Table 2, the false positive rate of the detection method in this example was 10%. As shown in Table 3, the test strips of this example do not have any cross-reactions with organophosphorus pesticides such as methamidophos, parathion, water amine thiophos and phorate. The result shows that the colloidal gold chromatography detection device for detecting the persistent phosphorus has relatively good specificity, and can accurately detect the persistent phosphorus in the vegetable and fruit samples, so that the persistent phosphorus residues in the vegetable and fruit samples can be rapidly detected.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.
Claims (9)
1. The immune hapten of the monocrotophos is characterized in that the structure of the immune hapten is shown as a formula (I):
，（Ⅰ）。
2. the coating hapten of the monocrotophos is characterized in that the structure is shown as a formula (II):
，（Ⅱ）。
3. a method of preparing an immune hapten of claim 1 or a coated hapten of claim 2, comprising the steps of:
S1, performing halogen substitution reaction on tert-butyl acetoacetate and N-chlorosuccinimide to obtain an intermediate 1, wherein the structural formula of the intermediate 1 is shown in a reaction formula (III):
Formula (III);
S2, carrying out Perkow rearrangement reaction on the intermediate 1 and trimethyl phosphite under heating in dichloroethane to obtain an intermediate 2, wherein the structural formula of the intermediate 2 is shown in a reaction formula (IV):
formula (IV);
s3, reacting the intermediate 2 with trifluoroacetic acid to remove tert-butyl groups to obtain an intermediate 3, wherein the structural formula of the intermediate 3 is shown in a reaction formula (V):
formula (V);
S4, reacting the intermediate 3 with N-hydroxysuccinimide to prepare first active ester, wherein the first active ester reacts with 4-aminobutyric acid under an alkaline condition to obtain the immune hapten MCP-H-1 of the monocrotophos, and the reaction formula is shown in the following formula (VI):
Formula (VI);
S5, reacting the intermediate 3 with N-hydroxysuccinimide under the action of a condensing agent to prepare a second active ester, wherein the second active ester reacts with 4-aminomethylphenylacetic acid under an alkaline condition to obtain the coating hapten MCP-H-2 of the monocrotophos, and the reaction formula is shown as the following formula (VII):
formula (VII).
4. The long-acting phosphorus antigen is characterized by comprising a long-acting phosphorus immune antigen and a long-acting phosphorus coating antigen, wherein the long-acting phosphorus immune antigen is a conjugate of the immune hapten of the long-acting phosphorus and carrier protein of claim 1, the long-acting phosphorus coating antigen is a conjugate of the coating hapten of the long-acting phosphorus and the carrier protein of claim 2, and the carrier protein is bovine serum albumin, human serum albumin, chicken egg serum albumin or hemocyanin.
5. Use of an immune hapten of claim 1 or a coated hapten of claim 2 or a phosphorus antigen of claim 4 for non-disease diagnosis in immunological detection of phosphorus.
6. The phosphorus-based antibody is characterized in that the phosphorus-based antibody is prepared from the phosphorus-based immune antigen of claim 4 through animal immunization, and the phosphorus-based antibody is a phosphorus-based monoclonal antibody.
7. Use of the antibody of claim 6 for non-disease diagnosis in immunological detection of monocrotophos.
8. The utility model provides a long-acting phosphorus colloidal gold chromatography detection device which characterized in that, includes test paper and reaction cup, the test paper includes the reaction membrane, the reaction membrane is equipped with detection zone and matter control district, the detection zone is coated with the long-acting phosphorus coating antigen of claim 4, contain the long-acting phosphorus antibody of claim 6 of colloidal gold mark in the reaction cup.
9. A method for detecting the persistent phosphorus in a sample, which is characterized in that the method adopts the colloidal gold chromatography detection device for the persistent phosphorus according to claim 8 to detect the persistent phosphorus in the sample, wherein the sample is an agricultural product.
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