CN110981791A - Paraquat derivative, preparation method thereof and paraquat detection reagent - Google Patents
Paraquat derivative, preparation method thereof and paraquat detection reagent Download PDFInfo
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Abstract
The invention discloses a paraquat derivative for detecting the content of paraquat in a human biological sample and a preparation method thereof. The series of anti-paraquat specific antibodies developed by utilizing the novel paraquat derivative can be used for preparing various paraquat immunological detection reagents with high sensitivity, strong specificity and good detection effect. The invention also provides a preparation method and a corresponding using method of the 3 paraquat immunoassay reagents. The 3 paraquat immunodetection methods provided by the invention are convenient to operate, rapid in detection, accurate in result, high in sensitivity and strong in specificity, and can be used for quantitatively detecting the content of paraquat in a human blood sample. The method overcomes the defects of complex operation, low automation degree and the like of the paraquat detection method in the prior art, and can effectively guide clinical individualized and reasonable medication.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a paraquat derivative, a preparation method thereof and a paraquat detection reagent.
Background
The chemical name of paraquat is 1, 1-dimethyl-4, 4-bipyridine cation salt, and the chemical structural formula is shown as formula VIII:
of the formula VIII
Paraquat is a quick herbicide, has contact action and certain systemic action, can be quickly absorbed by green tissues of plants to wither, has obvious killing power on the green plants, has no action on non-green tissues, is quickly combined with soil in the soil to be passivated, and has no effect on roots of the plants, perennial underground stems and perennial roots. Paraquat belongs to a highly toxic pesticide, has great toxicity to human bodies, does not have a specific antidote, has stimulation and corrosion effects on skin mucosa, can cause systemic poisoning after being orally taken for 3 g, can cause multiple system damages after being systemically poisoned, particularly causes changes of pulmonary congestion, bleeding, edema, transparent membrane formation and degeneration, hyperplasia, fibrosis and the like after being severely damaged by lung, can cause damage to liver and kidney and involvement of systems and organs such as circulation, nerves, blood, gastrointestinal tract, bladder and the like, and has extremely high death rate after being orally taken for poisoning. Paraquat is currently banned or severely restricted from use in more than 20 countries. The symptoms in the gold period of paraquat poisoning treatment are not obvious and are easy to misdiagnose or ignore.
The toxicity mechanism of paraquat is not clarified at present, most scholars think that paraquat is an electron receptor, can be actively transported by I-type and II-type lung cells and taken into cells, acts on redox reaction of the cells, and is activated into oxygen free radicals in the cells to be the basis of the toxicity effect of the cells, and the formed excessive super-oxidized anion free radicals, hydrogen peroxide and the like can cause the membrane lipid peroxidation of the cells of lung, liver and other tissues and organs, thereby causing the damage of the tissues and organs of multiple systems. The determination of the content of paraquat in a clinical plasma sample plays an important role in clinical treatment and prognosis prediction of paraquat poisoning patients.
The traditional paraquat test method mainly comprises the following steps: high performance liquid chromatography, reversed phase high performance liquid chromatography, and the like, which are complex to operate, slow in speed and high in cost. The paraquat specific series antibodies developed by utilizing the novel paraquat derivatives can be used for preparing various paraquat immunoassay reagents with high sensitivity, strong specificity and good detection effect, and can effectively make up for the defects of the traditional method. The 3 paraquat detection methods disclosed by the invention are high in detection speed, result accuracy and sensitivity, can be used for quantitative detection of clinical paraquat poisoning patient plasma samples, and have an important significance for improving the success rate of treatment of paraquat poisoning patients. Compared with the traditional method, the immunodetection method provided by the invention has the advantages of simplicity and convenience in operation, rapidness and quickness in detection, accurate result, low cost and the like, is favorable for future clinical popularization and application, and particularly has good application prospect for primary hospitals lacking expensive instruments.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical scheme adopted by the invention is as follows:
the paraquat derivative is a new synthetic substance which does not exist in nature, and has a structural formula shown as a formula I:
formula I.
Secondly, a synthetic method of the paraquat derivative is provided, which is different from the conventional synthetic method, has a good synthetic effect, and remarkably improves the synthetic efficiency of the paraquat derivative, and the specific synthetic route is as follows:
thirdly, providing a paraquat homogeneous enzyme immunoassay reagent, wherein the detection reagent consists of an R1 reagent and an R2 reagent, the R1 reagent comprises an anti-paraquat specific antibody 1 and an R1 buffer solution, and the R2 reagent comprises a paraquat glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution; the paraquat specific antibody 1 is an antibody produced by immunizing an experimental animal with paraquat chicken gamma globulin immunogen; the paraquat chicken gamma globulin immunogen is formed by coupling the paraquat derivative and chicken gamma globulin, and the structural formula of the paraquat derivative is shown as a formula II:
formula II;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the R1 buffer solution contains an enzyme substrate, coenzyme, bovine serum albumin and a Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is nicotinamide adenine dinucleotide oxidation type;
the paraquat glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling the paraquat derivative and glucose-6-phosphate dehydrogenase; the structural formula is shown as formula III:
formula III;
the R2 buffer solution is Tris buffer solution containing bovine serum albumin.
Fourthly, the preparation method of the paraquat homogeneous enzyme immunoassay reagent comprises the following steps:
(1) sequentially adding 0.25% bovine serum albumin, 50mmol/L glucose-6-phosphate and 50mmol/L nicotinamide adenine dinucleotide oxidized form into 50mmol/L Tris buffer solution, stirring and dissolving to prepare R1 buffer solution, adding an anti-paraquat specific antibody 1 into the R1 buffer solution according to the volume ratio of 1: 500-1: 5000, uniformly mixing, and adjusting the pH value to 8.0 by using 6 mol/L hydrochloric acid to prepare an R1 reagent;
(2) adding 0.25% bovine serum albumin into 100mmol/L Tris buffer solution, stirring and dissolving to prepare R2 buffer solution, adding paraquat glucose-6-phosphate dehydrogenase labeled conjugate into the R2 buffer solution according to the volume ratio of 1: 1000-1: 8000, mixing uniformly, and adjusting the pH value to 7.6 by using 6 mol/L hydrochloric acid to prepare R2 reagent;
the preparation method of the anti-paraquat specific antibody 1 comprises the following steps:
1) diluting the gamma globulin immunogen of the paraquat chicken to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
2) performing immune injection on an experimental animal by using a Freund's adjuvant method, extracting blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 1;
the preparation method of the paraquat chicken gamma globulin immunogen comprises the following steps:
① dissolving 100-300mg of gamma globulin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
②, dissolving 50-500mg of paraquat derivative by using 1.0-5.0ml of organic solvent A, activating by using a coupling activator, carrying out coupling reaction with the carrier protein solution prepared in the step ①, and dialyzing and purifying after the reaction is finished to obtain the immunogenic paraquat chicken gamma globulin immunogen;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
weighing glucose-6-phosphate dehydrogenase, dissolving the glucose-6-phosphate dehydrogenase in phosphate buffer solution with the pH value of 8.0 at room temperature to enable the final concentration of the glucose-6-phosphate dehydrogenase to be 2-6 mg/mL, and preparing enzyme solution;
dissolving the paraquat derivative by using the organic solvent A to enable the final concentration of the paraquat derivative to be 10-50mg/mL, activating by using a coupling activating agent, carrying out coupling reaction with the enzyme solution prepared in the step 1, and dialyzing and purifying after the reaction is finished to obtain a paraquat glucose-6-phosphate dehydrogenase labeled conjugate;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
The application method of the paraquat homogeneous enzyme immunoassay reagent comprises the following operation steps:
firstly, adding a calibrator and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding R2 reagent, mixing, keeping the temperature at 37 deg.C for 5-10 min, detecting at dominant wavelength of 340 nm/sub-wavelength of 405nm, continuously monitoring absorbance change rate within 3 min, and making calibration curve by full-automatic biochemical analyzer;
adding a sample to be detected and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding an R2 reagent, uniformly mixing, keeping the temperature at 37 ℃ for 5-10 minutes, detecting the dominant wavelength at 340 nm/sub-wavelength at 405nm, continuously monitoring the absorbance change rate within 3 minutes, and automatically calculating the content of paraquat in the sample to be detected by a full-automatic biochemical analyzer according to the calibration curve prepared in the step (I);
the reagent R1 and the reagent R2 are used according to the volume ratio of 1: 1-4: 1; the sample to be detected is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
Sixthly, providing a paraquat enzyme-linked immunosorbent (ELISA) detection reagent, wherein the detection reagent contains: anti-paraquat specific antibody 2, paraquat horseradish peroxidase labeled conjugate and reaction substrate;
the paraquat specific antibody 2 is an antibody produced by immunizing an experimental animal with paraquat bovine thyroglobulin immunogen; the paraquat bovine thyroglobulin immunogen is formed by coupling the paraquat derivative and bovine thyroglobulin, and the structural formula of the paraquat derivative is shown as a formula IV:
a formula IV;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the paraquat horseradish peroxidase labeled conjugate is formed by coupling the paraquat derivative and horseradish peroxidase; the structural formula is shown as formula V:
formula V;
the reaction substrate is 3,3',5,5' -tetramethyl benzidine;
the preparation method of the anti-paraquat specific antibody 2 comprises the following steps:
A. diluting the paraquat thyroglobulin immunogen to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
B. performing immune injection on the experimental animal by using a Freund's adjuvant method, extracting the blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 2;
the preparation method of the paraquat thyroglobulin immunogen comprises the following steps:
a. dissolving 100-100 mg of bovine thyroglobulin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
b. dissolving 50-500mg of paraquat derivative by using 1.0-5.0ml of organic solvent A, activating by using a coupling activator, carrying out coupling reaction with the carrier protein solution prepared in the step a, and dialyzing and purifying after the reaction is finished to obtain the immunogenic paraquat thyroglobulin immunogen;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat horseradish peroxidase labeled conjugate comprises the following steps:
(a) weighing horseradish peroxidase, dissolving in phosphate buffer solution with pH of 8.0 at room temperature to make final concentration of the solution be 2-6 mg/mL, and making into enzyme solution;
(b) dissolving the paraquat derivative by using the organic solvent A to enable the final concentration of the paraquat derivative to be 10-50mg/mL, activating by using a coupling activating agent, carrying out coupling reaction with the enzyme solution prepared in the step (a), and dialyzing and purifying after the reaction is finished to obtain a paraquat horseradish peroxidase labeled conjugate;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
Seventhly, the use method of the paraquat ELISA detection reagent comprises the following operation steps:
diluting anti-paraquat specific antibody 2 with phosphate buffer solution according to the proportion of 1: 1000-1: 20000 to prepare antibody solution, coating the antibody solution on a 96-well enzyme-linked plate according to the dosage of 100 mu L/well, and standing overnight at 4 ℃;
(ii) after washing 3 times with phosphate buffer, adding 200. mu.L/well of 0.5% bovine thyroglobulin solution, blocking overnight at 4 ℃, and washing 3 times with phosphate buffer;
(iii) adding 20 mu L/hole of standard substance and sample to be detected;
(iv) adding 100 mu L/hole of paraquat horseradish peroxidase labeled conjugate with working concentration;
(v) incubating for 30 min at room temperature and washing the plate 5 times with phosphate buffer;
(vi) adding 100. mu.L of 3,3',5,5' -tetramethylbenzidine per well, and incubating at room temperature for 30 minutes;
(vii) terminating the reaction by adding 100. mu.L of sulfuric acid having a concentration of 2mol/L to each well;
(viii) measuring the absorbance at a wavelength of 450nm by using a microplate reader;
(ix) preparing a calibration curve according to the light absorption values of the calibrators with different concentrations, and calculating the content of paraquat in the sample to be detected according to the calibration curve and the light absorption values of the sample to be detected;
the sample to be detected is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
Eighthly, providing a paraquat latex enhanced immunoturbidimetry detection reagent, wherein the detection reagent comprises: reagents L1 and L2;
the L1 reagent consists of an anti-paraquat specific antibody 3, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of paraquat-human serum albumin complex coated polystyrene latex particles, buffer solution with pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the anti-paraquat specific antibody 3 is an antibody produced by immunizing an experimental animal with paraquat hemocyanin immunogen; the paraquat hemocyanin immunogen is formed by coupling the paraquat derivative and hemocyanin, and the structural formula of the paraquat derivative is shown as a formula VI:
formula VI;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the paraquat-human serum albumin complex is formed by coupling the paraquat derivative and human serum albumin, and the structural formula of the complex is shown as a formula VII:
formula VII;
the diameter range of the polystyrene latex particles is 50-250 nm;
the buffer solution is any one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, Tris-HCl buffer solution or barbital buffer solution;
the coagulant is any one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is any one of sodium azide, thimerosal, phenol or ethylmercuric sodium thiosulfate.
Ninth, the preparation method of the paraquat latex enhanced immunoturbidimetry detection reagent comprises the following steps:
dissolving 0.5mg/mL of anti-paraquat specific antibody 3 in 50mmol/L phosphate buffer solution with pH =8.0, then adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% of sodium chloride, 0.1% -0.5% of Tween-20, 1.0% -5.0% of glycerol, 0.1% -1.0% of ethylenediamine tetraacetic acid, 0.5% -2.5% of PEG-4000 and 0.01% -0.1% of sodium azide by mass fraction, stirring uniformly, adjusting pH =7.5, and preparing an L1 reagent;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and 150nm in diameter into 4.5mL of 0.05mol/L MES buffer solution with pH =6.2, then adding 5mg carbodiimide, reacting at 37 ℃ for 1 hour to prepare latex particle solution, diluting 0.5mg of paraquat-human serum albumin complex with 4.5mL of 0.05mol/L borate buffer solution with pH =9.2, immediately adding the diluted solution into the latex particle solution, reacting at 37 ℃ for 12 hours, then adding 1mL of 0.1mol/L glycine buffer solution with pH =8.5, stirring for 2 hours, centrifuging after the reaction is ended to remove supernatant, washing the precipitate with 10mL of 50mmol/L Tris-HCl buffer solution with pH =8.0 for 3 times, diluting with 25mL of 50mmol/L glycine buffer solution with pH =8.5 to prepare latex suspension, and finally adding bovine serum albumin with mass fraction of 0.5% -2.5%, 0.25 to 1.0 percent of sodium chloride, 0.1 to 0.5 percent of tween-20, 1.0 to 5.0 percent of glycerol, 0.1 to 1.0 percent of ethylenediamine tetraacetic acid and 0.01 to 0.1 percent of sodium azide, and the mixture is stirred uniformly to prepare an L2 reagent;
the preparation method of the anti-paraquat specific antibody 3 comprises the following steps:
(A) diluting the paraquat hemocyanin immunogen to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
(B) performing immune injection on the experimental animal by using a Freund's adjuvant method, extracting the blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 3;
the preparation method of the paraquat hemocyanin immunogen comprises the following steps:
dissolving 100-300mg of hemocyanin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
dissolving 50-500mg of paraquat derivative by using 1.0-5.0ml of organic solvent A, activating by using a coupling activator, carrying out coupling reaction with the carrier protein solution prepared in the step (a), and dialyzing and purifying after the reaction is finished to obtain immunogenic paraquat hemocyanin immunogen;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat-human serum albumin complex comprises the following steps:
diluting 10mg of human serum albumin with 5mL of 0.1mol/L phosphate buffer solution with pH =7.8, then adding 100mg of the paraquat derivative, adding 50mg of the coupling activator, reacting at 4 ℃ for 12 hours, and dialyzing with 0.1mol/L phosphate buffer solution with pH =7.8 at 4 ℃ for 18 hours to obtain a paraquat-human serum albumin complex;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
The using method of the paraquat latex enhanced immunoturbidimetry detection reagent is basically the same as that of the paraquat homogeneous enzyme immunoassay reagent.
The calibrator consists of paraquat with the concentration of 0ng/ml, 40ng/ml, 80ng/ml, 160ng/ml, 320ng/ml and 640ng/ml respectively, sodium chloride with the mass fraction of 0.1-1.0%, bovine serum albumin with the mass fraction of 0.2-2.0%, ethylenediamine tetraacetic acid with the mass fraction of 0.25-1.5%, sodium azide with the mass fraction of 0.01-0.1% and Tris-HCl buffer solution with the pH of 50mmol/L = 7.2.
The series of paraquat-resistant specific antibodies developed by utilizing the novel paraquat derivative can be used for preparing various paraquat immunoassay reagents with high sensitivity, strong specificity and good detection effect. The invention also provides a preparation method and a corresponding using method of the 3 paraquat immunoassay reagents. The 3 paraquat immunodetection methods provided by the invention are convenient to operate, rapid in detection, accurate in result, high in sensitivity and strong in specificity, and can be used for quantitatively detecting the content of paraquat in samples such as human serum and blood plasma. The method overcomes the defects of complex operation, low automation degree and the like of the paraquat detection method in the prior art, and can effectively guide clinical individualized and reasonable medication.
Drawings
FIG. 1 is a calibration curve of a homogeneous enzyme immunoassay reagent for paraquat;
FIG. 2 is a calibration curve of the detection reagent for paraquat ELISA;
FIG. 3 is a calibration curve of a paraquat latex enhanced immunoturbidimetric assay reagent.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, which are simplified schematic drawings illustrating only the basic structure of the present invention and showing only the constitution related to the present invention, and detailed embodiments. Unless otherwise specified, reagents, instruments, equipment, consumables used in the following examples are all available from a regular distributor.
EXAMPLE 1 Synthesis of Paraquat derivatives
The chemical structure of the paraquat derivative is shown as formula I:
formula I.
The specific route of the synthesis method of the paraquat derivative is as follows:
the specific synthetic steps are as follows:
1. synthesis of Compound 2
10 g of Compound 1 was dissolved in 100mL of DMF, and then 20 mL of DIEA and 8.5 g of MeI were added to prepare a reaction solution, and the reaction solution was stirred at room temperature overnight, 200 mL of purified water was added to the mixture after the reaction, followed by filtration, and the cake obtained by the filtration was dried in vacuo to obtain 10 g of Compound 2 as a white solid with a yield of 91%.
2. Synthesis of paraquat derivatives
5.0 g of Compound 2 was dissolved in 50mL of DMF, and then 5.3 g of 5-bromovaleric acid was added at 0 ℃ to prepare a reaction solution, the reaction solution was stirred at room temperature overnight, and then the reaction solution was evaporated under reduced pressure and passed through SiO2The obtained 2.3 g paraquat derivative is yellow solid after purification by flash column chromatography, and the yield is 29%.
Example 2 preparation of Paraquat homogeneous enzyme immunoassay reagent
The preparation method of the paraquat homogeneous enzyme immunoassay reagent comprises the following specific steps:
(1) sequentially adding 0.25% bovine serum albumin, 50mmol/L glucose-6-phosphate and 50mmol/L nicotinamide adenine dinucleotide oxidized form into 50mmol/L Tris buffer solution, stirring and dissolving to prepare R1 buffer solution, adding an anti-paraquat specific antibody 1 into the R1 buffer solution according to the volume ratio of 1: 1500, uniformly mixing, and adjusting the pH value to 8.0 by using 6 mol/L hydrochloric acid to prepare an R1 reagent;
(2) adding 0.25% bovine serum albumin into 100mmol/L Tris buffer solution, stirring and dissolving to prepare R2 buffer solution, adding paraquat glucose-6-phosphate dehydrogenase labeled conjugate into the R2 buffer solution according to the volume ratio of 1: 3000, uniformly mixing, and adjusting the pH value to 7.6 by using 6 mol/L hydrochloric acid to prepare the R2 reagent.
The preparation method of the anti-paraquat specific antibody 1 comprises the following steps:
1) diluting the gamma globulin immunogen of the paraquat chicken to a final concentration of 2.5mg/mL by using a phosphate buffer solution;
2) performing immune injection on the experimental animal rabbit by using a Freund's adjuvant method, extracting blood of the rabbit after 5 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 1;
the preparation method of the paraquat chicken gamma globulin immunogen comprises the following steps:
① dissolving 200mg of gamma globulin in 50ml of 0.2 mol/L phosphate buffer solution with pH of 8.5 to obtain carrier protein solution;
② dissolving 300mg of paraquat derivative described in example 1 in 3.0ml of dimethyl sulfoxide, activating with N-hydroxysuccinimide, performing coupling reaction with the carrier protein solution prepared in step ①, and dialyzing and purifying after the reaction is finished to obtain the immunogenic paraquat chicken gamma globulin immunogen.
The preparation method of the paraquat glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
<1> glucose-6-phosphate dehydrogenase was weighed and dissolved in a phosphate buffer solution having a pH of 8.0 at room temperature to a final concentration of 4.5mg/mL to prepare an enzyme solution;
<2> the paraquat derivative described in example 1 was dissolved using dimethylformamide to a final concentration of 25mg/mL, activated by N, N' -dicyclohexylcarbodiimide, and subjected to a coupling reaction with the enzyme solution prepared in step <1>, and after the reaction was completed, the paraquat glucose-6-phosphate dehydrogenase-labeled conjugate was obtained by dialysis purification.
EXAMPLE 3 preparation of Paraquat calibrator
Respectively adding the pure paraquat powder into 5 parts of Tris-HCl buffer solution with the concentration of 50mmol/L and the pH =7.2, stirring and dissolving until the final concentrations are respectively 0ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL and 640ng/mL, then respectively adding sodium chloride with the mass fraction of 0.5%, bovine serum albumin with the mass fraction of 1.0%, ethylenediaminetetraacetic acid with the mass fraction of 0.75% and sodium azide with the mass fraction of 0.05% into each solution, and uniformly stirring to obtain the paraquat calibrator (with the concentration of 6).
Example 4 Paraquat homogeneous enzyme immunoassay reagent calibration curve preparation and quality control experiment
1. Preparing a homogeneous enzyme immunoassay calibration curve:
placing an R1 reagent, an R2 reagent and a calibrator into a Merrill BS480 full-automatic biochemical analyzer, and then setting reaction parameters of the biochemical analyzer, wherein the specific parameters are detailed in Table 1; in the actual operation process, the volume ratio of the R1 reagent to the R2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a homogeneous enzyme immunoassay calibration curve is automatically obtained by a biochemical analyzer, as shown in figure 1.
TABLE 1 Merrill BS480 fully automated Biochemical Analyzer reaction parameters
| Name of item | Paraquat (Paraquat) |
| R1 reagent | 180µl |
| R2 reagent | 60µl |
| Sample size | 20µl |
| Calibration method | End point method |
| Dominant wavelength | 340nm |
| Sub-wavelength | 405nm |
| Reaction time | 10 minutes |
| Incubation time | 8 minutes |
| Reaction direction | Rise up |
| Results | ng/ml |
| Accuracy of results | 0.01 |
| Fitting method | Line graph |
| Concentration of standard substance | 0ng/mL、40ng/mL、80ng/mL、160ng/mL、320ng/mL、640ng/mL |
2. Quality control experiment:
dissolving pure paraquat powder in methanol to prepare 1mg/mL stock solution, diluting the stock solution in healthy human plasma without paraquat until the final concentrations are respectively 0.00, 20.00, 200.00 and 600.00ng/mL, and preparing blank, low, medium and high-concentration quality control samples. The quality control samples are measured by using the paraquat homogeneous enzyme immunoassay method, the content of paraquat in each quality control sample is calculated according to the homogeneous enzyme immunoassay calibration curve prepared in the step 1, each quality control sample is repeatedly measured for 10 times, and the detection results and data analysis are detailed in table 2.
TABLE 2 Paraquat homogeneous enzyme immunoassay reagent detection results and data analysis
| Quality control sample | Blank space | Is low in | In | Height of |
| Sample concentration (ng/ml) | 0.00 | 20.00 | 200.00 | 600.00 |
| Test 1 | 0.00 | 20.52 | 205.03 | 593.27 |
| Test 2 | 0.01 | 20.76 | 202.74 | 595.51 |
| Test 3 | 0.00 | 19.89 | 203.91 | 608.89 |
| Test 4 | 0.00 | 20.45 | 207.30 | 612.21 |
| Test 5 | 0.00 | 20.05 | 196.82 | 589.60 |
| Test 6 | 0.02 | 20.17 | 199.40 | 614.98 |
| Test 7 | 0.00 | 20.33 | 205.79 | 606.66 |
| Test 8 | 0.00 | 19.96 | 198.58 | 592.50 |
| Test 9 | 0.00 | 20.27 | 200.87 | 590.45 |
| Test 10 | 0.00 | 19.88 | 197.33 | 608.99 |
| Mean value (ng/ml) | 0.00 | 20.23 | 201.78 | 601.31 |
| Standard Deviation (SD) | / | 0.29 | 3.71 | 9.90 |
| Precision (CV%) | / | 1.43 | 1.84 | 1.65 |
| Recovery (%) | / | 101.15 | 100.89 | 100.22 |
The experimental results show that: the CV values of the paraquat content in the quality control samples with different concentrations are lower than 5%, and the recovery rates are between 95% and 105%, which shows that the precision of the paraquat content in the biological sample measured by the paraquat homogeneous enzyme immunoassay reagent is high, and the result is accurate.
Example 5 preparation of Key Components in Paraquat ELISA assay reagents
1. A method for preparing an anti-paraquat specific antibody 2, comprising the steps of:
A. diluting the paraquat thyroglobulin immunogen to a final concentration of 3.0mg/mL by using a phosphate buffer solution;
B. performing immunization injection on a sheep of an experimental animal by using a Freund's adjuvant method, extracting blood of the sheep after 4 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 2;
the preparation method of the paraquat thyroglobulin immunogen comprises the following steps:
a. dissolving 150mg of bovine thyroglobulin in 75ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
b. dissolving 250mg of paraquat derivative described in example 1 in 2.5ml of dimethylformamide, activating by tributylamine, performing coupling reaction with the carrier protein solution prepared in the step a, and performing dialysis purification after the reaction is finished to obtain immunogenic paraquat thyroglobulin immunogen;
2. the preparation method of the paraquat horseradish peroxidase labeled conjugate comprises the following steps:
(a) weighing horseradish peroxidase, dissolving in phosphate buffer solution with pH of 8.0 at room temperature to make final concentration of 3.5mg/mL, and making into enzyme solution;
(b) dissolving the paraquat derivative described in example 1 in isopropanol to a final concentration of 35mg/mL, activating with N, N' -dicyclohexylcarbodiimide, performing a coupling reaction with the enzyme solution prepared in step (a), and dialyzing and purifying after the reaction is finished to obtain a paraquat horseradish peroxidase-labeled conjugate.
Example 6 evaluation experiment of Paraquat ELISA test reagent Properties
1. Preparation of Paraquat ELISA detection calibration curve
Diluting an anti-paraquat specific antibody 2 by phosphate buffer solution according to the proportion of 1: 10000 to prepare antibody solution, coating the antibody solution on a 96-hole enzyme-linked plate according to the dosage of 100 mu L/hole, and standing overnight at 4 ℃;
(ii) after washing 3 times with phosphate buffer, adding 200. mu.L/well of 0.5% bovine thyroglobulin solution, blocking overnight at 4 ℃, and washing 3 times with phosphate buffer;
(iii) adding 20. mu.L/well of standard
(iv) adding 100 mu L/hole of paraquat horseradish peroxidase labeled conjugate with working concentration;
(v) incubating for 30 min at room temperature and washing the plate 5 times with phosphate buffer;
(vi) adding 100. mu.L of 3,3',5,5' -tetramethylbenzidine per well, and incubating at room temperature for 30 minutes;
(vii) terminating the reaction by adding 100. mu.L of sulfuric acid having a concentration of 2mol/L to each well;
(viii) measuring the absorbance at a wavelength of 450nm by using a microplate reader;
(ix) preparing a calibration curve based on absorbance values of calibrators of different concentrations, as shown in FIG. 2.
2. Quality control experiment
Dissolving pure paraquat powder in methanol to prepare 1mg/mL stock solution, diluting the stock solution in healthy human plasma without paraquat until the final concentrations are respectively 0.00, 20.00, 200.00 and 600.00ng/mL, and preparing blank, low, medium and high-concentration quality control samples. And measuring the absorbance values of the blank, low, medium and high-concentration quality control samples at 450nm by using the paraquat ELISA detection method. And (3) comparing the calibration curve of the paraquat ELISA detection shown in FIG. 2, calculating the content of paraquat in each quality control sample, repeating the measurement for 3 times for each quality control sample, calculating the recovery rate according to the measurement result, and obtaining the detection data in Table 3.
TABLE 3 Paraquat ELISA test reagent performance evaluation data
| Quality control sample | Blank space | Is low in | In | Height of |
| Sample concentration (ng/mL) | 0.00 | 20.00 | 200.00 | 600.00 |
| Test 1 | 0.00 | 20.37 | 205.26 | 592.36 |
| Test 2 | 0.00 | 20.72 | 207.04 | 583.50 |
| Test 3 | 0.00 | 20.15 | 193.85 | 615.29 |
| Mean value (ng/mL) | 0.00 | 20.41 | 202.05 | 597.05 |
| Recovery (%) | / | 102.07 | 101.03 | 99.51 |
The experimental results show that: the recovery rate of the paraquat content in samples with different concentrations, which are measured by the paraquat ELISA detection reagent, is in the range of 95-105%, which shows that the accuracy of the paraquat content in a biological sample measured by the paraquat ELISA detection reagent is higher.
Example 7 preparation of Paraquat latex enhanced Immunoturbidimetric assay reagent
The preparation method of the paraquat latex enhanced immunoturbidimetry detection reagent comprises the following steps:
dissolving 0.5mg/mL anti-paraquat specific antibody 3 in 50mmol/L phosphate buffer solution with pH =8.0, then adding 1.25% by mass of bovine serum albumin, 0.75% by mass of sodium chloride, 0.25% by mass of Tween-20, 2.75% by mass of glycerol, 0.5% by mass of ethylenediamine tetraacetic acid, 1.25% by mass of PEG-4000 and 0.03% by mass of sodium azide, stirring uniformly, adjusting pH =7.5, and preparing a reagent L1;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and 150nm in diameter into 4.5mL of 0.05mol/L MES buffer with pH =6.2, then adding 5mg carbodiimide, reacting at 37 ℃ for 1 hour to prepare a latex particle solution, diluting 0.5mg of paraquat-human serum albumin complex with 4.5mL of 0.05mol/L borate buffer with pH =9.2, immediately adding the diluted solution into the latex particle solution, reacting at 37 ℃ for 12 hours, then adding 1mL of 0.1mol/L glycine buffer with pH =8.5, stirring for 2 hours, centrifuging after the reaction is ended to remove the supernatant, washing the precipitate with 10mL of 50mmol/L Tris-HCl buffer with pH =8.0 for 3 times, diluting with 25mL of 50mmol/L glycine buffer with pH =8.5 to prepare a latex suspension, and finally adding bovine serum albumin with the mass fraction of 1.25%, 0.55% of sodium chloride, 0.25% of tween-20, 3.0% of glycerol, 0.45% of ethylenediamine tetraacetic acid and 0.03% of sodium azide, and stirring uniformly to prepare an L2 reagent;
the preparation method of the anti-paraquat specific antibody 3 comprises the following steps:
(A) diluting the paraquat hemocyanin immunogen to a final concentration of 2.5mg/mL by using a phosphate buffer solution;
(B) performing immune injection on an experimental animal horse by using a Freund's adjuvant method, extracting blood of the horse after 6 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 3;
the preparation method of the paraquat hemocyanin immunogen comprises the following steps:
dissolving 200mg of hemocyanin in 60ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
dissolving 250mg of paraquat derivative by using 2.5ml of dimethylformamide, activating by using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, carrying out coupling reaction with the carrier protein solution prepared in the step (a), and dialyzing and purifying after the reaction is finished to obtain immunogenic paraquat hemocyanin immunogen;
the preparation method of the paraquat-human serum albumin complex comprises the following steps:
10mg of human serum albumin was diluted with 5mL of 0.1mol/L phosphate buffer solution with pH =7.8, and then 100mg of the above paraquat derivative was added, and 50mg of N-hydroxysuccinimide was added to react at 4 ℃ for 12 hours, followed by dialysis with 0.1mol/L phosphate buffer solution with pH =7.8 at 4 ℃ for 18 hours, to obtain a paraquat-human serum albumin complex.
Example 8 Paraquat latex enhanced immunoturbidimetric assay reagent calibration Curve preparation and quality control experiment
1. Preparing a calibration curve of the latex enhanced immunoturbidimetric assay reagent:
placing R1 reagent, R2 reagent and calibrator in an Olympus AU480 full-automatic biochemical analyzer, and setting reaction parameters of the biochemical analyzer, wherein the detailed parameters are shown in Table 4; in the actual operation process, the volume ratio of the R1 reagent to the R2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a latex enhanced immunoturbidimetric detection calibration curve is automatically obtained by a biochemical analyzer, as shown in FIG. 3.
TABLE 4 reaction parameters of Orlinbas AU480 fully automatic biochemical analyzer
| Name of item | Paraquat (Paraquat) |
| R1 reagent | 240µl |
| R2 reagent | 80µl |
| Sample size | 30µl |
| Calibration method | End point method |
| Dominant wavelength | 570nm |
| Sub-wavelength | 412nm |
| Reaction ofTime of day | 8 minutes |
| Incubation time | 5 minutes |
| Reaction direction | Descend |
| Results | ng/ml |
| Accuracy of results | 0.01 |
| Fitting method | Line graph |
| Concentration of standard substance | 0ng/mL、40ng/mL、80ng/mL、160ng/mL、320ng/mL、640ng/mL |
2. Quality control experiment:
dissolving pure paraquat powder in methanol to prepare 1mg/mL stock solution, diluting the stock solution in healthy human plasma without paraquat until the final concentrations are respectively 0.00, 20.00, 200.00 and 600.00ng/mL, and preparing blank, low, medium and high-concentration quality control samples. The latex enhanced turbidimetric immunoassay method is utilized to measure the quality control samples, the content of paraquat in each quality control sample is calculated according to the latex enhanced turbidimetric immunoassay calibration curve prepared in the step 1, each quality control sample is repeatedly measured for 10 times, and the detection results and data analysis are detailed in table 5.
TABLE 5 Paraquat latex enhanced immunoturbidimetric reagent test results and data analysis
| Quality control sample | Blank space | Is low in | In | Height of |
| Sample concentration (ng/ml) | 0.00 | 20.00 | 200.00 | 600.00 |
| Test 1 | 0.00 | 20.34 | 202.53 | 586.08 |
| Test 2 | 0.00 | 20.88 | 205.99 | 593.41 |
| Test 3 | 0.01 | 19.75 | 203.36 | 602.29 |
| Test 4 | 0.00 | 20.28 | 206.51 | 608.46 |
| Test 5 | 0.00 | 20.09 | 196.83 | 589.81 |
| Test 6 | 0.00 | 20.52 | 194.54 | 610.98 |
| Test 7 | 0.00 | 20.16 | 202.70 | 603.00 |
| Test 8 | 0.00 | 19.89 | 198.00 | 595.28 |
| Test 9 | 0.01 | 20.55 | 200.01 | 598.76 |
| Test 10 | 0.00 | 19.61 | 207.55 | 611.90 |
| Mean value (ng/ml) | 0.00 | 20.21 | 201.80 | 600.00 |
| Standard Deviation (SD) | / | 0.39 | 4.37 | 8.90 |
| Precision (CV%) | / | 1.93 | 2.17 | 1.48 |
| Recovery (%) | / | 101.05 | 100.90 | 100.00 |
The experimental results show that: the CV values of the paraquat content in the quality control samples with different concentrations are lower than 5%, and the recovery rates are between 95% and 105%, which shows that the precision of the paraquat content in the biological sample measured by the paraquat latex enhanced immunoturbidimetry detection reagent is high, and the result is accurate.
In light of the foregoing description of the preferred embodiments of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. A paraquat derivative is characterized by having a structural formula shown as a formula I:
formula I.
2. A method for synthesizing paraquat derivatives according to claim 1, wherein the specific route of the method is as follows:
3. a paraquat homogeneous enzyme immunoassay reagent, which consists of an R1 reagent and an R2 reagent, wherein the R1 reagent comprises an anti-paraquat specific antibody 1 and an R1 buffer solution, and the R2 reagent comprises a paraquat glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution; the paraquat specific antibody 1 is an antibody produced by immunizing an experimental animal with paraquat chicken gamma globulin immunogen; the paraquat chicken gamma globulin immunogen is formed by coupling paraquat derivative and chicken gamma globulin according to claim 1, and the structural formula of the paraquat derivative is shown as a formula II:
formula II;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the R1 buffer solution contains an enzyme substrate, coenzyme, bovine serum albumin and a Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is nicotinamide adenine dinucleotide oxidation type;
the paraquat glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling paraquat derivative and glucose-6-phosphate dehydrogenase as defined in claim 1; the structural formula is shown as formula III:
formula III;
the R2 buffer solution is Tris buffer solution containing bovine serum albumin.
4. A method for preparing the paraquat homogeneous enzyme immunoassay reagent according to claim 3, wherein the method comprises the following steps:
(1) sequentially adding 0.25% bovine serum albumin, 50mmol/L glucose-6-phosphate and 50mmol/L nicotinamide adenine dinucleotide oxidized form into 50mmol/L Tris buffer solution, stirring and dissolving to prepare R1 buffer solution, adding an anti-paraquat specific antibody 1 into the R1 buffer solution according to the volume ratio of 1: 500-1: 5000, uniformly mixing, and adjusting the pH value to 8.0 by using 6 mol/L hydrochloric acid to prepare an R1 reagent;
(2) adding 0.25% bovine serum albumin into 100mmol/L Tris buffer solution, stirring and dissolving to prepare R2 buffer solution, adding paraquat glucose-6-phosphate dehydrogenase labeled conjugate into the R2 buffer solution according to the volume ratio of 1: 1000-1: 8000, mixing uniformly, and adjusting the pH value to 7.6 by using 6 mol/L hydrochloric acid to prepare R2 reagent;
the preparation method of the anti-paraquat specific antibody 1 comprises the following steps:
1) diluting the gamma globulin immunogen of the paraquat chicken to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
2) performing immune injection on an experimental animal by using a Freund's adjuvant method, extracting blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 1;
the preparation method of the paraquat chicken gamma globulin immunogen comprises the following steps:
① dissolving 100-300mg of gamma globulin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
② dissolving 50-500mg of paraquat derivative as claimed in claim 1 in 1.0-5.0ml of organic solvent A, activating by coupling activator and performing coupling reaction with carrier protein solution prepared in step ①, and dialyzing and purifying after reaction to obtain immunogenic gamma globulin immunogen of paraquat chicken;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
weighing glucose-6-phosphate dehydrogenase, dissolving the glucose-6-phosphate dehydrogenase in phosphate buffer solution with the pH value of 8.0 at room temperature to enable the final concentration of the glucose-6-phosphate dehydrogenase to be 2-6 mg/mL, and preparing enzyme solution;
dissolving the paraquat derivative of claim 1 by using the organic solvent A to obtain a final concentration of 10-50mg/mL, activating by using a coupling activating agent, carrying out coupling reaction with the enzyme solution prepared in the step <1>, and dialyzing and purifying after the reaction is finished to obtain a paraquat glucose-6-phosphate dehydrogenase labeled conjugate;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
5. The use of the homogeneous enzyme immunoassay reagent for paraquat according to claim 3, comprising the following steps:
firstly, adding a calibrator and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding R2 reagent, mixing, keeping the temperature at 37 deg.C for 5-10 min, detecting at dominant wavelength of 340 nm/sub-wavelength of 405nm, continuously monitoring absorbance change rate within 3 min, and making calibration curve by full-automatic biochemical analyzer;
adding a sample to be detected and an R1 reagent into a full-automatic biochemical analyzer, uniformly mixing, and incubating for 3-5 minutes at 37 ℃; adding an R2 reagent, uniformly mixing, keeping the temperature at 37 ℃ for 5-10 minutes, detecting the dominant wavelength at 340 nm/sub-wavelength at 405nm, continuously monitoring the absorbance change rate within 3 minutes, and automatically calculating the content of paraquat in the sample to be detected by a full-automatic biochemical analyzer according to the calibration curve prepared in the step (I);
the reagent R1 and the reagent R2 are used according to the volume ratio of 1: 1-4: 1; the sample to be detected is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
6. A paraquat ELISA detection reagent, comprising: anti-paraquat specific antibody 2, paraquat horseradish peroxidase labeled conjugate and reaction substrate;
the paraquat specific antibody 2 is an antibody produced by immunizing an experimental animal with paraquat bovine thyroglobulin immunogen; the paraquat bovine thyroglobulin immunogen is formed by coupling paraquat derivative and bovine thyroglobulin in claim 1, and the structural formula of the paraquat derivative is shown as a formula IV:
a formula IV;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the paraquat horseradish peroxidase labeled conjugate is formed by coupling paraquat derivatives and horseradish peroxidase according to claim 1; the structural formula is shown as formula V:
formula V;
the reaction substrate is 3,3',5,5' -tetramethyl benzidine;
the preparation method of the anti-paraquat specific antibody 2 comprises the following steps:
A. diluting the paraquat thyroglobulin immunogen to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
B. performing immune injection on the experimental animal by using a Freund's adjuvant method, extracting the blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 2;
the preparation method of the paraquat thyroglobulin immunogen comprises the following steps:
a. dissolving 100-100 mg of bovine thyroglobulin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
b. dissolving 50-500mg of paraquat derivative as defined in claim 1 in 1.0-5.0ml of organic solvent A, activating by coupling activator and carrying out coupling reaction with the carrier protein solution prepared in step a, and dialyzing and purifying after the reaction is finished to obtain immunogenic paraquat thyroglobulin immunogen;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat horseradish peroxidase labeled conjugate comprises the following steps:
(a) weighing horseradish peroxidase, dissolving in phosphate buffer solution with pH of 8.0 at room temperature to make final concentration of the solution be 2-6 mg/mL, and making into enzyme solution;
(b) dissolving the paraquat derivative of claim 1 by using the organic solvent A to make the final concentration of the paraquat derivative be 10-50mg/mL, activating the paraquat derivative by using a coupling activating agent, performing coupling reaction with the enzyme solution prepared in the step (a), and performing dialysis purification after the reaction is finished to obtain a paraquat horseradish peroxidase labeled conjugate;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
7. The method for using the paraquat ELISA detection reagent as claimed in claim 6, is characterized by comprising the following operation steps:
diluting anti-paraquat specific antibody 2 with phosphate buffer solution according to the proportion of 1: 1000-1: 20000 to prepare antibody solution, coating the antibody solution on a 96-well enzyme-linked plate according to the dosage of 100 mu L/well, and standing overnight at 4 ℃;
(ii) after washing 3 times with phosphate buffer, adding 200. mu.L/well of 0.5% bovine thyroglobulin solution, blocking overnight at 4 ℃, and washing 3 times with phosphate buffer;
(iii) adding 20 mu L/hole of standard substance and sample to be detected;
(iv) adding 100 mu L/hole of paraquat horseradish peroxidase labeled conjugate with working concentration;
(v) incubating for 30 min at room temperature and washing the plate 5 times with phosphate buffer;
(vi) adding 100. mu.L of 3,3',5,5' -tetramethylbenzidine per well, and incubating at room temperature for 30 minutes;
(vii) terminating the reaction by adding 100. mu.L of sulfuric acid having a concentration of 2mol/L to each well;
(viii) measuring the absorbance at a wavelength of 450nm by using a microplate reader;
(ix) preparing a calibration curve according to the light absorption values of the calibrators with different concentrations, and calculating the content of paraquat in the sample to be detected according to the calibration curve and the light absorption values of the sample to be detected;
the sample to be detected is any one of serum, plasma, urine, saliva, tissue fluid or cerebrospinal fluid.
8. A paraquat latex enhanced immunoturbidimetry detection reagent is characterized by comprising: reagents L1 and L2;
the L1 reagent consists of an anti-paraquat specific antibody 3, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of paraquat-human serum albumin complex coated polystyrene latex particles, buffer solution with pH =8.0, bovine serum albumin, sodium chloride, Tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the anti-paraquat specific antibody 3 is an antibody produced by immunizing an experimental animal with paraquat hemocyanin immunogen; the paraquat hemocyanin immunogen is formed by coupling paraquat derivative and hemocyanin according to claim 1, and the structural formula of the paraquat derivative is shown as a formula VI:
formula VI;
the experimental animal is any one of rabbit, goat, mouse, sheep, guinea pig or horse;
the paraquat-human serum albumin complex is formed by coupling paraquat derivative as claimed in claim 1 and human serum albumin, and the structural formula of the paraquat-human serum albumin complex is shown as a formula VII:
formula VII;
the diameter range of the polystyrene latex particles is 50-250 nm;
the buffer solution is any one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, Tris-HCl buffer solution or barbital buffer solution;
the coagulant is any one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is any one of sodium azide, thimerosal, phenol or ethylmercuric sodium thiosulfate.
9. The preparation method of the paraquat latex enhanced turbidimetric immunoassay reagent as claimed in claim 8, wherein the preparation method comprises the following steps:
dissolving 0.5mg/mL of anti-paraquat specific antibody 3 in 50mmol/L phosphate buffer solution with pH =8.0, then adding 0.5% -2.5% of bovine serum albumin, 0.25% -1.0% of sodium chloride, 0.1% -0.5% of Tween-20, 1.0% -5.0% of glycerol, 0.1% -1.0% of ethylenediamine tetraacetic acid, 0.5% -2.5% of PEG-4000 and 0.01% -0.1% of sodium azide by mass fraction, stirring uniformly, adjusting pH =7.5, and preparing an L1 reagent;
adding 0.5mg of polystyrene latex particles with carboxyl groups on the surface and 150nm in diameter into 4.5mL of 0.05mol/L MES buffer solution with pH =6.2, then adding 5mg carbodiimide, reacting at 37 ℃ for 1 hour to prepare latex particle solution, diluting 0.5mg of paraquat-human serum albumin complex with 4.5mL of 0.05mol/L borate buffer solution with pH =9.2, immediately adding the diluted solution into the latex particle solution, reacting at 37 ℃ for 12 hours, then adding 1mL of 0.1mol/L glycine buffer solution with pH =8.5, stirring for 2 hours, centrifuging after the reaction is ended to remove supernatant, washing the precipitate with 10mL of 50mmol/L Tris-HCl buffer solution with pH =8.0 for 3 times, diluting with 25mL of 50mmol/L glycine buffer solution with pH =8.5 to prepare latex suspension, and finally adding bovine serum albumin with mass fraction of 0.5% -2.5%, 0.25 to 1.0 percent of sodium chloride, 0.1 to 0.5 percent of tween-20, 1.0 to 5.0 percent of glycerol, 0.1 to 1.0 percent of ethylenediamine tetraacetic acid and 0.01 to 0.1 percent of sodium azide, and the mixture is stirred uniformly to prepare an L2 reagent;
the preparation method of the anti-paraquat specific antibody 3 comprises the following steps:
(A) diluting the paraquat hemocyanin immunogen to a final concentration of 0.5-5.0mg/mL by using a phosphate buffer solution;
(B) performing immune injection on the experimental animal by using a Freund's adjuvant method, extracting the blood of the animal after 3-8 times of injection, and separating and purifying antiserum to obtain an anti-paraquat specific antibody 3;
the preparation method of the paraquat hemocyanin immunogen comprises the following steps:
dissolving 100-300mg of hemocyanin in 10-100ml of phosphate buffer solution with the concentration of 0.2 mol/L and the pH value of 8.5 to prepare carrier protein solution;
dissolving 50-500mg of paraquat derivative as claimed in claim 1 in 1.0-5.0ml of organic solvent A, activating by coupling activator and carrying out coupling reaction with the carrier protein solution prepared in the step < a >, and dialyzing and purifying after the reaction is finished to obtain immunogenic paraquat hemocyanin immunogen;
the organic solvent A is any one of dimethyl sulfoxide, dimethylformamide, isopropanol, methanol or ethanol;
the preparation method of the paraquat-human serum albumin complex comprises the following steps:
diluting 10mg of human serum albumin with 5mL of 0.1mol/L phosphate buffer solution with pH =7.8, then adding 100mg of paraquat derivative according to claim 1, adding 50mg of coupling activator, reacting at 4 ℃ for 12 hours, and dialyzing with 0.1mol/L phosphate buffer solution with pH =7.8 at 4 ℃ for 18 hours to obtain paraquat-human serum albumin complex;
the coupling activator is any one of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, tributylamine, glutaraldehyde, a diisocyanate compound or dihalogenated dinitrobenzene.
10. The calibrator according to any one of claims 5 or 7, wherein the calibrator consists of paraquat at a concentration of 0ng/ml, 40ng/ml, 80ng/ml, 160ng/ml, 320ng/ml, 640ng/ml, sodium chloride at a mass fraction of 0.1-1.0%, bovine serum albumin at a mass fraction of 0.2-2.0%, ethylenediaminetetraacetic acid at a mass fraction of 0.25-1.5%, sodium azide at a mass fraction of 0.01-0.1%, Tris-HCl buffer at a pH of 50mmol/L =7.2, respectively.
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