CN107607721B - Chlorpromazine magnetic immunochemiluminescence detection kit and application thereof - Google Patents
Chlorpromazine magnetic immunochemiluminescence detection kit and application thereof Download PDFInfo
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Abstract
The invention provides a chlorpromazine magnetic immune chemiluminescence detection kit, which comprises: the kit comprises an enzyme-labeled antigen, an enzyme-labeled antigen diluent, a magnetic-labeled antibody diluent, chlorpromazine series standard substance solutions, a chemiluminescent substrate A solution, a chemiluminescent substrate B solution, a complex solution and a washing solution, wherein the enzyme-labeled antigen is a chlorpromazine hapten marked by horseradish peroxidase, and the magnetic-labeled antibody is a chlorpromazine monoclonal antibody marked by immunomagnetic beads. The invention also discloses a method for detecting the chlorpromazine residual quantity in animal tissues and milk by using the kit matched with the chemiluminescence detector, and the method has higher sensitivity and specificity and shorter detection time for the chlorpromazine detection.
Description
Technical Field
The invention relates to a chemiluminescence detection kit and a detection method thereof, in particular to a magnetic immunity chemiluminescence detection kit for detecting chlorpromazine residual quantity in samples such as animal tissues, milk and the like, belonging to the field of immunological detection.
Background
Chlorpromazine (Chlorpromazine) is a phenothiazine drug, is a blocker of central dopamine receptors, acts on the central nervous system, and has various pharmacological effects such as antipsychotic, cooling, antiemetic, hypnotic, anesthesia and the like. Veterinary medicine is mainly used as sedative in clinical practice. In recent years, driven by benefits, chlorpromazine is added privately in feed and drinking water to achieve the effects of calming and hypnosis, so that the effects of fattening and promoting growth are indirectly achieved; or in the transportation process, in order to reduce weight loss and stress death in the way and prevent meat quality from decreasing, chlorpromazine is often used in large amount, and especially before slaughtering, the chlorpromazine is used in large amount, so that chlorpromazine can be left in livestock and poultry products.
Chlorpromazine is mainly metabolized by the liver in vivo and mostly eliminated from urine, but accumulation and residue in vivo are easy to generate due to slow excretion, and the residue can be remained in animal tissues for several months. The residual chlorpromazine in animal tissues enters human bodies through food chains, can cause leucopenia, agranulocytosis and other adverse reactions, and directly influences the physical health of people. Meanwhile, the product outlet is influenced due to the excessive residue, and huge economic loss is brought to the breeding industry. Therefore, the addition of chlorpromazine to feed (EC) No.17/97 was mandated by the European Union as early as 1997; the 176 th bulletin (2002) of department of agriculture in China also clearly indicates that: chlorpromazine is strictly prohibited to be added into animal feed and drinking water; ministry of agriculture publication No. 235 (2002) again states: the therapeutic effect of chlorpromazine is allowed but not detectable in animal products.
Driven by economic benefits, the phenomenon of illegal abuse of chlorpromazine is often prohibited, and the classical chromatographic method cannot meet the rapid detection requirement of a large number of samples on site, so that the appearance of portable and high-sensitivity rapid detection products is urgently needed. The immunoassay method is a rapid analysis method established by taking the specific reaction of an antigen and an antibody as a basic principle, and the technology is widely applied to the field of drug residue detection due to the high selectivity and sensitivity of the combination of the antigen and the antibody. Currently, the most commonly used immunodetection methods include enzyme-linked immunosorbent assay and colloidal gold immunochromatographic assay, but due to the low sensitivity and high false negative and false positive rates, the methods are gradually replaced by chemiluminescence immunodetection methods with high sensitivity, high accuracy and short detection time. The magnetic immunochemiluminescence detection reagent is matched with a magnetic immunochemiluminescence detector to detect the residual amount of the chlorpromazine in the animal-derived product, so that the full automation of the detection process is realized, the manual operation error is reduced, the sensitivity is high, the accuracy is high, the detection cost is low, and the magnetic immunochemiluminescence detection reagent is suitable for screening the residual amount of the chlorpromazine in a large batch of samples.
Disclosure of Invention
The invention aims to provide a chlorpromazine detection kit, which has higher sensitivity, specificity and accuracy when used for detecting the chlorpromazine residual quantity.
The invention also aims to provide a method for detecting chlorpromazine, which has higher sensitivity, specificity and accuracy when a chemiluminescence detector matched with the kit is used for detecting the residual amount of the chlorpromazine, realizes full-automatic detection, shortens the detection time and reduces the manual operation error.
The kit of the invention comprises: enzyme-labeled antigen, enzyme-labeled antigen diluent, magnetic-labeled antibody diluent, chlorpromazine series standard substance solution, chemiluminescence substrate A solution, chemiluminescence substrate B solution, complex solution and washing solution.
The enzyme-labeled antigen is a chlorpromazine hapten labeled by horse radish peroxidase, the magnetic labeled antibody is a chlorpromazine monoclonal antibody labeled by immunomagnetic beads, and the chlorpromazine monoclonal antibody is prepared by taking a conjugate obtained by coupling the chlorpromazine hapten and bovine serum albumin as an immunogen to immunize animals; the chlorpromazine hapten is obtained by reacting an N1- (5-chloro-2-iodobenzene) -N3 compound with 3-bromo-2-thiophenol to obtain an intermediate hydroxy chlorpromazine and then reacting the intermediate hydroxy chlorpromazine with iodoacetaldehyde, and the molecular structural formula of the chlorpromazine hapten is as follows:
the surface of the immunomagnetic bead contains-OH, -COOH or-NH2An active group.
The chemiluminescence substrate A solution is a trihydroxymethyl aminomethane solution containing luminol and p-cresol, and the chemiluminescence substrate B solution is a trihydroxymethyl aminomethane solution containing citric acid and anhydrous Na2HPO4And CO (NH)2)2·H2O2An aqueous solution of (a).
The concentration of the chlorpromazine series standard solution is 0 mug/L, 0.05 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L and 4.05 mug/L respectively.
The enzyme-labeled antigen diluent is Na with pH of 7.2-7.63PO4Concentration of 0.01mol/L, NaCl concentration of 0.25mol/L of buffer solution.
The magnetic labeling antibody diluent is Na with pH of 7.2-7.63PO4The concentration of the buffer solution is 0.01mol/L, NaCl and the concentration of the buffer solution is 0.25 mol/L.
The double solution is phosphate buffer solution with pH 7.0 and 0.1 mol/L.
The washing solution is pH7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide and 0.1-0.3 mol/L of phosphate buffer solution.
The kit is matched with a chemiluminescence detector to detect the residual amount of chlorpromazine in animal tissues and milk samples.
The invention also provides a method for detecting the residual amount of chlorpromazine by using the chemiluminescence detector matched with the kit, which comprises the following steps:
1) diluting an enzyme-labeled antigen and an enzyme-labeled antigen diluent according to a volume ratio of 1: 10-1: 20, and filling the diluted enzyme-labeled antigen and the enzyme-labeled antigen diluent into a chemiluminescence detector enzyme-labeled antigen working solution container;
2) diluting a magnetic standard antibody and a magnetic standard antibody diluent according to a volume ratio of 1: 10-1: 20, and putting the diluted magnetic standard antibody and the magnetic standard antibody diluent into a chemiluminescence detector magnetic standard antibody working solution container;
3) respectively absorbing 30-60 mu L of enzyme-labeled antigen working solution, 30-60 mu L of sample solution to be detected and 30-60 mu L of magnetic label antibody working solution by a chemiluminescence detector, sequentially adding the solutions into a reaction cup storage device, reacting for 15min at room temperature, carrying out magnetic separation for 2-4 min by a cleaning device, removing supernatant, and cleaning the compound precipitate for 3-5 times by 300-500 mu L of cleaning solution;
4) and (3) putting the separated compound into a measurement dark box, adding 50 mu L of each of the chemiluminescence substrate A liquid and the chemiluminescence substrate B liquid, detecting the emitted relative light intensity, enabling the content of the chlorpromazine in the sample to have a negative correlation with the relative light intensity, and calculating the residual amount of the chlorpromazine through a relative light intensity standard curve.
The chemiluminescence detector used in the analysis and test method comprises a power supply circuit, a reaction cup storage device, a sample arm, a reagent storage device, a reagent arm, a movable refrigeration device, a cleaning device, an automatic injection pump, a low-light-level detector, and Windows control software with a computer and a Chinese interface, can perform functions of data entry, result summarization, quality control, result storage, result query and the like, can complete programming of various analysis modes, report results quantitatively or qualitatively, automatically generate, store and update functions, and automatically correct standard curves at two points.
The invention has the following beneficial effects:
1) the kit has higher sensitivity and specificity, and the detection sensitivity of the kit on the chlorpromazine can reach 0.05 mu g/L.
2) The kit provided by the invention is matched with a chemiluminescence detector to detect the residual amount of chlorpromazine in a sample, so that the full automation of the detection process is realized, the manual operation error is reduced, the detection time is short, and the detection of the residual amount of chlorpromazine in the sample can be completed in only 20 min.
Drawings
FIG. 1: chlorpromazine hapten synthesis scheme
FIG. 2: standard curve diagram of magnetic immune chemiluminescence detection kit
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: preparation of concrete components of chlorpromazine magnetic immune chemiluminescence detection kit
1. Synthesis and identification of chlorpromazine hapten (the synthetic route is shown in figure 1)
A.1.0g of N1- (5-chloro-2-iodobenzene) -N3 compound is dissolved in anhydrous N, N-Dimethylformamide (DMF), 0.8g of cuprous iodide is added, 0.3g of sodium hydride is added, 0.66g of 3-bromo-2-thiophenol is added, the mixture is heated in an oil bath, the reaction is stopped for 4 hours at 100 ℃, the mixture is cooled to room temperature, water is added, ethyl acetate is added for extraction, rotary evaporation is carried out and evaporation is carried out to dryness, the mixture is applied to a silica gel column, and petroleum ether/ethyl acetate (v/v, 10/1) is eluted and separated to obtain 0.9g of intermediate hydroxychloropiperazine, and the yield is 91.84%;
B. taking 0.9g of hydroxyl chlorpromazine, adding acetonitrile for dissolving and clarifying, adding 0.54g of iodoacetaldehyde and 0.43g of potassium carbonate, stirring for 3 hours at 50 ℃, stopping reaction, adding water, extracting with ethyl acetate, evaporating to dryness to obtain a crude product, and recrystallizing with dichloromethane/n-hexane (v/v, 1/1) to obtain 0.98g of acetaldehyde chlorpromazine hapten product, wherein the yield is 96.74%.
Taking the hapten, performing nuclear magnetic resonance hydrogen spectrum identification,1H-NMR(CDCl3,300MHz)：9.71(1H,s,CHO)，7.10(2H,dd,ArH)，6.76(2H,dd,ArH)，6.26(1H,dd,ArH)，5.50(2H,dd,CH2)，3.93(2H,dd,CH2)，2.46(2H,dd,CH2)，2.26(2H,s,CH3)，1.67(2H,dd,CH2). Wherein, the chemical shift is 9.71 and 5.50 are resonance absorption peaks of aldehyde group hydrogen on the spacer arm, and the existence of the peaks proves the success of hapten synthesis.
2. Preparation of enzyme-labeled antigen
Taking 14mg of chlorpromazine hapten, adding 0.3mL of ethanol for dissolving and clarifying to obtain solution A; dissolving horseradish peroxidase (HRP)50mg in 0.1mol/L carbonate buffer solution to obtain solution B; dripping the A solution into the B solution, stirring overnight at 4 ℃, dialyzing and purifying 0.02mol/L PB for three days, changing the solution three times per day to obtain the enzyme-labeled antigen, and subpackaging and storing at-20 ℃.
3. Preparation of immunogens
HRP is replaced by Bovine Serum Albumin (BSA), and the preparation method is the same as the above, so that the immunogen is obtained.
4. Preparation of chlorpromazine monoclonal antibody
(1) Obtaining hybridoma cells
1) First immunization: chlorpromazine hapten-BSA conjugate (immunogen) and an equal amount of Freund's complete adjuvant are fully emulsified, and 6-week-old Balb/c mice are injected subcutaneously, wherein each mouse is 0.2 mL;
2) two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;
3) after one week of last boosting immunization, measuring the titer and inhibition in fundus venous blood sampling, and performing the following last immunization when the titer reaches more than 1: 10000: injecting 0.1mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;
4) and (4) measuring cell supernatant by adopting an indirect enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the chlorpromazine monoclonal antibody, preparing the hybridoma cell in a logarithmic growth phase into a cell suspension by using a freezing medium, subpackaging in a freezing tube, and storing in liquid nitrogen for a long time.
(2) Preparation of monoclonal antibodies
1) Cell recovery: taking out the cryopreserving tube of the chlorpromazine monoclonal antibody hybridoma cell strain, immediately putting the cryopreserving tube into a water bath at 37 ℃ for medium-speed melting, centrifugally removing a cryopreserving liquid, and transferring the cryopreserving liquid into a culture bottle for culture;
2) preparing ascites and purifying antibody by injecting 0.5mL of sterilized paraffin oil into the abdominal cavity of Balb/c mouse (8 weeks old) by in vivo induction method, and injecting hybridoma cells 5 × 10 into the abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying by octanoic acid-saturated ammonium sulfate method, determining purity by SDS-PAGE electrophoresis, subpackaging with small bottles, and storing at-20 deg.C.
5. Preparation of magnetic labeled antibody
The process takes carboxyl magnetic beads with the particle size of 2.8 mu m as a carrier, the tail ends of the carboxyl magnetic beads have reactive carboxyl groups, and after the carboxyl magnetic beads are combined and treated by an activating agent EDC-NHS, the activated magnetic beads can be coupled with a chlorpromazine monoclonal antibody, and the specific steps are as follows:
(1) cleaning: 100 μ L of carboxyl magnetic beads (purchased from DYNAL, particle size of 2.8 μm, content of 0.15eq/g) were placed in a centrifuge tube, washed 2 times with 100 μ L of MES solution containing 0.05% Tween-20, pH5.0, 25mmol/L, and the supernatant was removed after magnetic separation;
(2) and (3) activation: respectively preparing 50mmol/L EDC solution and NHS solution by using 25mmol/L MES solution with pH value of 5.0 stored at 4 ℃; respectively adding 50 μ L of newly prepared EDC and NHS solution into a centrifuge tube filled with magnetic beads, mixing uniformly by vortex, activating at room temperature for 30min, removing supernatant after magnetic separation, and washing with (1) MES solution for 2-3 times;
(3) coupling: dissolving chlorpromazine monoclonal antibody into 60 mu L of MES solution with pH of 5.0 and 25mmol/L, adjusting the total volume to 100 mu L with the MES solution, gently adding the solution into activated magnetic beads, and coupling at room temperature for 30min or 4 ℃ for 2h, wherein the magnetic beads are kept in a uniform mixing state;
(4) and (3) sealing: removing supernatant after magnetic separation, adding 100 μ L TRIS solution with pH of 7.4, reacting for 15min, and sealing magnetic beads;
(5) and (3) storage: removing supernatant after magnetic separation, washing the sealed magnetic beads with 100 μ L TRIS solution containing 0.1% -0.3% BSA and 0.1% Tween-20 for 3-5 times, removing supernatant after magnetic separation, and re-dissolving the magnetic beads in a solution containing 0.1% -0.5% BSA, 0.01% -0.1% Tween-20, and 0.02% NaN3The TRIS solution (concentration: 10mg/mL) was stored at 2-8 ℃ until use.
Example 2: construction of chlorpromazine magnetic immunity chemiluminescence detection kit
The chlorpromazine magnetic immunochemiluminescence detection kit is constructed and comprises the following components:
(1) horseradish peroxidase-labeled chlorpromazine hapten;
(2) the enzyme-labeled antigen diluent is pH 7.2-7.6 and Na3PO4A buffer solution with the concentration of 0.01mol/L, NaCl and the concentration of 0.25 mol/L;
(3) chlorpromazine monoclonal antibody marked by immunomagnetic beads;
(4) the dilution liquid of the magnetic labeled antibody is pH 7.2-7.6 and Na3PO4A buffer solution with the concentration of 0.01mol/L, NaCl and the concentration of 0.25 mol/L;
(5) the concentration of the chlorpromazine series standard solution is 0 mug/L, 0.05 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L and 4.05 mug/L respectively;
(6) the chemiluminescence substrate A solution is a trihydroxymethyl aminomethane solution containing luminol and p-cresol, and the chemiluminescence substrate B solution is a trihydroxymethyl aminomethane solution containing citric acid and anhydrous Na2HPO4And CO (NH)2)2·H2O2An aqueous solution of (a);
(7) the compound solution is phosphate buffer solution with pH 7.0 and 0.1 mol/L;
(8) the washing solution is pH7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide and 0.1-0.3 mol/L of phosphate buffer solution.
Example 3: detection of chlorpromazine residual quantity in sample
1. Sample pretreatment
(1) Animal tissue (chicken, pork, fish, shrimp) samples: weighing 2.0g + -0.05 g homogenized tissue sample into a 50mL polystyrene centrifuge tube, adding 2mL 2.5% trichloroacetic acid and 6mL acetone respectively, shaking for 5min with a shaker, mixing, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; transferring 2mL of the supernatant into a 50mL polystyrene centrifuge tube, adding 2mL of 2mol/L sodium hydroxide solution and 4.5mL of n-hexane respectively, shaking for 5min with a shaker, mixing, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; transferring 3mL of the upper organic phase into a 10mL clean dry glass tube, and drying the upper organic phase in a water bath nitrogen flow at 50-60 ℃ (122-; adding 0.5mL of the complex solution, and vortexing for 2min by using a vortex instrument to dissolve the dried residue; 50 μ L was taken for analysis.
(2) Milk sample: transferring 1mL fresh milk sample into a 50mL polystyrene centrifuge tube, adding 1mL 10% trichloroacetic acid, vortexing for 2min with a vortex instrument, adding 6mL acetone, vigorously shaking for 5min with a shaker, mixing, and centrifuging for 5min at 3000g room temperature (20-25 deg.C/68-77 deg.F); transferring 2mL of the supernatant into a 50mL polystyrene centrifuge tube, adding 2mL of 2mol/L sodium hydroxide solution and 4.5mL of n-hexane respectively, shaking for 5min with a shaker, mixing, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; transferring 3mL of the upper organic phase into a 10mL clean dry glass tube, and drying the upper organic phase in a water bath nitrogen flow at 50-60 ℃ (122-; adding 0.5mL of the complex solution, and vortexing for 2min by using a vortex instrument to dissolve the dried residue; 50 μ L was taken for analysis.
2. Detection with a kit
Diluting an enzyme-labeled antigen and an enzyme-labeled antigen diluent according to a volume ratio of 1: 10-1: 20, and filling the diluted enzyme-labeled antigen and the enzyme-labeled antigen diluent into a chemiluminescence detector enzyme-labeled antigen working solution container; diluting a magnetic standard antibody and a magnetic standard antibody diluent according to a volume ratio of 1: 10-1: 20, and putting the diluted magnetic standard antibody and the magnetic standard antibody diluent into a chemiluminescence detector magnetic standard antibody working solution container; setting the position on a sample rack for each sample/standard product, and inputting sample information and a test project name to be detected; putting the sample tube/standard sample tube on a set sample frame, sequentially sucking 50 mu L of enzyme-labeled antigen working solution, 50 mu L of sample/standard product to be detected and 50 mu L of magnetic standard antibody working solution by a chemiluminescence detector, adding the mixture into a reaction cup storage device, uniformly mixing, reacting for 15min at room temperature, carrying out magnetic separation for 4min by a cleaning device, removing supernatant, cleaning the precipitate of the compound by using a cleaning solution for 3-5 times, adding 50 mu L of each of a chemiluminescence substrate A solution and a chemiluminescence substrate B solution, detecting relative light intensity (RLU) emitted by the mixture, enabling the content of chlorpromazine in the sample to be in a negative correlation with the RLU, and calculating the residual amount of chlorpromazine by combining the RLU with a standard curve method.
3. Analysis of detection results
Dividing the RLU mean (RLU) of the standard solutions obtained for each concentration by the RLU value (RLU) of the first standard solution (0 standard)0) And multiplied by 100% to obtain the relative luminescence intensity (%). A standard curve is drawn by taking the logarithmic value of the chlorpromazine standard substance concentration (μ g/L) as the abscissa and the relative luminous intensity as the ordinate, as shown in FIG. 2. The relative luminous intensity of the sample solution is calculated by the same method, and the chlorpromazine content corresponding to each sample can be read from the standard curve.
Example 4: quality evaluation of chlorpromazine magnetic immune chemiluminescence detection kit
1. Detection limit
The detection is carried out on 20 parts of blank pork, chicken, fish, shrimp and milk samples respectively, the concentration corresponding to each relative luminous intensity is found out from the standard curve, the average value of the 20 parts of sample concentration is added with 3 times of standard deviation to represent the detection limit, and as a result, the detection limit of the method on animal tissue samples is 0.1 mug/kg, and the detection limit on milk samples is 0.2 mug/L.
2. Sample precision and accuracy testing
The recovery rate is used as an accuracy evaluation index, and the relative standard deviation (RSD%) of the detection result of a sample with a certain concentration is repeatedly measured and used as a precision evaluation index. The calculation formula is as follows: recovery (%) — actual measurement/theoretical value × 100%, where theoretical value is the added concentration of the sample; relative standard deviation RSD% ═ SD/X × 100%, where SD is the standard deviation and X is the average of the measured data.
The method comprises the steps of performing addition recovery measurement on blank pork, chicken, fish and shrimp samples according to three chlorpromazine concentrations of 0.1 mu g/kg, 0.2 mu g/kg and 0.4 mu g/kg, performing addition recovery measurement on the blank milk samples according to three chlorpromazine concentrations of 0.2 mu g/L, 0.4 mu g/L and 0.8 mu g/L, performing 4 parallel measurement on each sample, performing measurement by using three different kits, and calculating the average recovery rate and precision of the samples, wherein the results are shown in Table 1.
TABLE 1 precision and accuracy tests
Adding chlorpromazine with three concentrations of 0.1, 0.2 and 0.4 mu g/kg to blank pork, chicken, fish and shrimp samples, and adding chlorpromazine with three concentrations of 0.2, 0.4 and 0.8 mu g/L to blank milk samples, wherein the average recovery rate is 70-110%; the relative standard deviation in each batch and between batches is less than 10 percent.
3. Specificity test
The drugs with similar structures and functions to chlorpromazine are selected for determination, the 50% inhibition concentration of each drug is obtained through a standard curve of each drug, the cross reaction rate of the kit to other drugs is calculated by the following formula, and the result is shown in table 2.
TABLE 2 Cross-reactivity test
| Name of drug | IC50 | Cross-reactivity (%) |
| Chlorpromazine | 0.145 | 100 |
| Promethazine | — | ＜1 |
| Acetylpromazine maleic acid | — | ＜1 |
Note: IC (integrated circuit)50Is "-" because the curve is distorted and deformed, it cannot be used to calculate a specific value.
4. Stability test
The storage condition of the kit is 2-8 ℃, and the maximum RLU value (zero standard), the 50% inhibition concentration and the actual measurement value of chlorpromazine addition of the kit are within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
Claims (5)
1. A chlorpromazine magnetic immune chemiluminescence detection kit comprises an enzyme-labeled antigen, an enzyme-labeled antigen diluent, a magnetic-labeled antibody diluent, chlorpromazine series standard substance solutions, a chemiluminescence substrate A solution, a chemiluminescence substrate B solution, a complex solution and a cleaning solution; the method is characterized in that: the enzyme-labeled antigen is a chlorpromazine hapten labeled by horse radish peroxidase, the magnetic labeled antibody is a chlorpromazine monoclonal antibody labeled by immunomagnetic beads, and the chlorpromazine monoclonal antibody is prepared by taking a conjugate obtained by coupling the chlorpromazine hapten and bovine serum albumin as an immunogen to immunize animals; the synthesis method of the chlorpromazine hapten comprises the following steps: dissolving 1.0g of N1- (5-chloro-2-iodobenzene) -N3 compound in anhydrous N, N-dimethylformamide, adding 0.8g of cuprous iodide, 0.3g of sodium hydride and 0.66g of 3-bromo-2-thiophenol, heating in an oil bath, reacting at 100 ℃ for 4 hours, stopping reaction, cooling to room temperature, adding water, extracting with ethyl acetate, evaporating to dryness, loading on a silica gel column, eluting with petroleum ether/ethyl acetate in a volume ratio of 10:1, and separating to obtain an intermediate, namely, hydroxychloropiperazine 0.9; taking 0.9g of hydroxyl chlorpromazine, adding acetonitrile for dissolving and clarifying, adding 0.54g of iodoacetaldehyde and 0.43g of potassium carbonate, stirring for 3 hours at 50 ℃, stopping reaction, adding water, extracting with ethyl acetate, evaporating to dryness to obtain a crude product, and recrystallizing with dichloromethane/n-hexane at a volume ratio of 1:1 to obtain 0.98g of an acetaldehyde chlorpromazine hapten, namely a chlorpromazine hapten, wherein the molecular structural formula of the acetaldehyde chlorpromazine hapten is as follows:
2. the kit of claim 1, wherein: the surface of the immunomagnetic bead contains-OH, -COOH or-NH2An active group.
3. The kit of claim 1, wherein: the chemiluminescence substrate A solution is a trihydroxymethyl aminomethane solution containing luminol and p-cresol, and the chemiluminescence substrate B solution is a trihydroxymethyl aminomethane solution containing citric acid and anhydrous Na2HPO4And CO (NH)2)2·H2O2An aqueous solution of (a).
4. The kit of claim 1, wherein: the kit is matched with a chemiluminescence detector to detect the residual amount of chlorpromazine.
5. A method for detecting chlorpromazine by using the kit of any one of claims 1 to 4, comprising the steps of:
1) diluting an enzyme-labeled antigen and an enzyme-labeled antigen diluent according to a volume ratio of 1: 10-1: 20, and filling the diluted enzyme-labeled antigen and the enzyme-labeled antigen diluent into a chemiluminescence detector enzyme-labeled antigen working solution container;
2) diluting a magnetic standard antibody and a magnetic standard antibody diluent according to a volume ratio of 1: 10-1: 20, and putting the diluted magnetic standard antibody and the magnetic standard antibody diluent into a chemiluminescence detector magnetic standard antibody working solution container;
3) respectively absorbing 30-60 mu L of enzyme-labeled antigen working solution, 30-60 mu L of sample solution to be detected and 30-60 mu L of magnetic label antibody working solution by a chemiluminescence detector, sequentially adding the solutions into a reaction cup storage device, reacting for 15min at room temperature, carrying out magnetic separation for 2-4 min by a cleaning device, removing supernatant, and cleaning the compound precipitate for 3-5 times by 300-500 mu L of cleaning solution;
4) and (3) putting the separated compound into a measurement dark box, adding 50 mu L of each of the chemiluminescence substrate A liquid and the chemiluminescence substrate B liquid, detecting the emitted relative light intensity, enabling the content of the chlorpromazine in the sample to have a negative correlation with the relative light intensity, and calculating the residual amount of the chlorpromazine through a relative light intensity standard curve.
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| CN104897895A (en) * | 2015-04-14 | 2015-09-09 | 北京勤邦生物技术有限公司 | Gentamycin magnetic immunochemiluminescence detection kit and application thereof |
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| CN101987836A (en) * | 2010-08-31 | 2011-03-23 | 华南农业大学 | Chlorpromazine hapten, artificial antigen, antibody as well as preparation method and application thereof |
| CN102928402A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting chlorpromazine, and applications thereof |
| CN104897895A (en) * | 2015-04-14 | 2015-09-09 | 北京勤邦生物技术有限公司 | Gentamycin magnetic immunochemiluminescence detection kit and application thereof |
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