CN114858767A - Fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots - Google Patents
Fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots Download PDFInfo
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Abstract
The invention relates to the technical field of immunodetection, in particular to a fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots. The invention provides a method for labeling a olaquindox antigen by CdTe quantum dots, which comprises the following steps: adding EDC and NHS into the olaquindox antigen solution, standing, then adding CdTe quantum dots, and standing for reaction; the particle size range of the CdTe quantum dots is 8-15 nm; a large number of groping experiments show that the olaquindox antigen can cover the surface defects of the CdTe quantum dots to form a fluorescent marker with relatively stable fluorescence performance, and the fluorescence intensity is increased by 500-1000.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots.
Background
Olaquindox (Olaquindox, Ola), an antibacterial growth promoter, is also known as quinamide alcohol, betaminox, and mefenox. The feed additive has the effects of promoting protein assimilation, improving feed conversion rate and excellent antibacterial effect, and is widely used for growth and antibacterial treatment of livestock such as pigs, cattle, sheep, horses, rabbits and the like, but bacterial drug resistance is increased easily if Ola is abused, and livestock meat polluted by Ola is possibly infected with drug-resistant bacteria if people eat the bacteria by mistake, so that health is harmed. Studies have shown severe organic damage to the liver and kidneys of animals; can also damage the immune organs of animals and inhibit the immune function of erythrocytes; can also induce the apoptosis of animal spermatogenic cells; modern toxicological studies indicate that Ola and its metabolite 3-methyl-quinoxaline-2-carboxylic acid have strong accumulative toxicity and teratogenicity effects. The european union has banned Ola from being used in edible animals in 1998; the rural part of agriculture in China stipulates that operation is stopped and Ola bulk drugs and various preparations are used from 5 months in 2019, but abuse conditions still exist in some aquaculture industries. In order to maintain the rights and interests of consumers, guarantee the physical health of people, strengthen the supervision and management on food safety, and develop more accurate, convenient and economic food safety detection methods, the research and development of the food safety detection methods are urgent.
The main detection method at present is liquid chromatography tandem mass spectrometry. The method is sensitive and high in accuracy, but the instrument is expensive, the pretreatment is complex, and professional technicians are required to operate the method. In recent years, quantum dot enzyme-linked immunoassay technology combining QDs labeling technology and ELISA can realize ultramicro measurement of specific targets (antigen molecules) such as heavy metals, pathogenic bacteria, biotoxin, pesticide and veterinary drug residues, chemical pollutants and the like, has the advantages of simplicity and convenience in operation, rapidness, accuracy, specificity, sensitivity, low price, high efficiency, simplicity in pretreatment, no need of special detection equipment and the like, is concerned by more and more researchers, and has great application value and bright prospect in the field of food safety detection. By combining with the immunoassay technical method, more efficient, simple and economical novel immunoassay methods can be established. However, no relevant research on detection of olaquindox by a fluorescence immunoassay method exists at present, so that the fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots is provided, the blank of detection of olaquindox by the fluorescence immunoassay method is filled, and powerful technical support is provided for further development of food safety detection.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots.
Therefore, the invention provides the following technical scheme:
a method for labeling a olaquindox antigen by CdTe quantum dots comprises the following steps: adding EDC and NHS into the olaquindox antigen solution, standing, then adding CdTe quantum dots, and standing for reaction; the particle size range of the CdTe quantum dots is 8-15 nm.
Optionally, 0.1-0.5mL of olaquindox antigen solution with the concentration of 0.1-0.8mg/mL is taken, 20-60 muL of EDC solution with the concentration of 0.05-0.30mol/L and 20-60 muL of NHS solution with the concentration of 0.05-0.30mol/L are added, standing is carried out for 8-20min at the temperature of 10-26 ℃, 0.05-0.5mL of CdTe quantum dot solution with the concentration of 0.001-0.005mol/L is added, buffer solution is added to reach 2-10mL of constant volume, and then the mixture is placed for 36-48h at the temperature of 0-6 ℃;
optionally, 0.2mL of olaquindox antigen solution with the concentration of 0.5mg/mL is taken, 50 muL of EDC solution with the concentration of 0.10mol/L and 50 muL of NHS solution with the concentration of 0.1mol/L are added, standing is carried out for 15min at the temperature of 10-26 ℃, then 0.1mL of CdTe quantum dot solution with the concentration of 0.001mol/L is added, buffer solution is added to the solution to reach the constant volume of 5mL, and then the solution is placed for 36-48h at the temperature of 0-6 ℃;
optionally, when the particle size of the CdTe quantum dots is 8nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.3mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 μ L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 μ L;
optionally, when the particle size of the CdTe quantum dot is 11nm, the concentration of the CdTe quantum dot solution is 0.004mol/L, the added volume is 0.20mL, the concentration of the olaquindox antigen solution is 0.20mg/mL, the added volume is 0.3mL, the concentration of the EDC solution is 0.2mol/L, the added volume is 40 μ L, the concentration of the NHS solution is 0.2mol/L, and the added volume is 40 μ L;
optionally, when the particle size of the CdTe quantum dot is 15nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.45mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 μ L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 μ L.
The application of the CdTe quantum dot-labeled olaquindox antigen obtained by the labeling method in detection of olaquindox is disclosed;
optionally, the use of olaquindox in the detection of animal meat or animal urine;
optionally, use in detecting pork, beef, mutton, horse meat, rabbit meat, pig urine, cow urine, sheep urine, horse urine or rabbit urine.
A fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots comprises the following steps:
coating: using a olaquindox antibody as a coating antigen, diluting the coating antigen with a coating buffer solution to obtain a coating solution, coating an ELISA plate, and washing;
and (3) sealing: sealing the coated ELISA plate with a sealing liquid, and washing;
drawing a standard curve: adding a series of olaquindox standard solutions with standard concentrations and a CdTe quantum dot labeled olaquindox antigen solution diluent into the closed enzyme label plate to perform indirect competitive reaction, simultaneously setting a control solution without olaquindox, washing the plate, detecting the fluorescence value, taking the lg value of the olaquindox standard solution concentration as a horizontal coordinate, taking the binding rate as a vertical coordinate, and drawing a standard curve; the CdTe quantum dot marked olaquindox antigen solution is marked by the method of any one of claims 1-2; the binding rate is multiplied by 100 percent of the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox;
and (3) determination: adding a sample to be detected and a CdTe quantum dot-labeled olaquindox antigen solution diluent into the closed enzyme label plate to perform indirect competitive reaction, washing the plate, detecting the fluorescence value, calculating the binding rate, and substituting the obtained binding rate into the standard curve to calculate the concentration of the sample to be detected;
optionally, the coating buffer solution is 0.01-0.05mol/L PBS (pH7.0-7.8), 0.005-0.03mol/L PBST (pH7.4-7.8), 0.005-0.05mol/L CBS (pH8.0-10.2) or 0.005-0.03mol/L Tris-HCl (pH7.8-9.6);
optionally, the coating buffer is 0.01mol/L PBS (pH7.4), 0.0lmol/L PBST (pH7.4), 0.05mol/L CBS (pH9.6) or 0.01mol/L Tris-HCl (pH8.0);
optionally, the coating buffer is 0.01mol/L Tris-HCl with pH value of 8.0;
optionally, the olaquindox antibody is used as a coating antigen, the concentration of the coating antigen is diluted to 2-5 mug/mL by using a coating buffer solution to obtain a coating solution, then an ELISA plate is added according to 20-50 microliter/hole, and the coating is carried out for 5-16h at the temperature of 2-10 ℃.
Optionally, the blocking solution is 0.1-2% v/v ovalbumin
Optionally, the blocking solution is 0.1% v/v, 0.5% v/v, 1% v/v or 2% v/vOVA;
optionally, the confining liquid is 0.5% v/v OVA;
optionally, adding blocking liquid into the coated enzyme label plate according to 20-50 microliters/hole, wherein the blocking condition is that the temperature is 10-20 ℃ and the time is 5-15 hours.
Optionally, in the step of drawing a standard curve, a series of olaquindox standard solutions with standard concentration and a dilution of olaquindox antigen solution marked by CdTe quantum dots are obtained by diluting with a first dilution, and the concentration range of the series of olaquindox standard solutions with standard concentration is 10 -6 –10 -1 mg/mL, wherein the dilution multiple of the CdTe quantum dot marked olaquindox antigen solution is 4-16 times by volume; the first diluent is a buffer solution containing 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS, and the pH value is 7.4;
optionally, adding 20-50 microliters/hole of a series of olaquindox standard solutions with standard concentrations and 20-50 microliters/hole of a CdTe quantum dot labeled olaquindox antigen solution diluent into the closed enzyme label plate, adjusting the pH value to 7.4, and reacting at 37 ℃ for at least 150 min;
optionally, adding a series of olaquindox standard solutions with standard concentration 50 microliter/pore and olaquindox antigen solution diluent marked by CdTe quantum dots 50 microliter/pore into the sealed enzyme label plate, adjusting the pH value to 7.4, reacting at 37 ℃ for at least 150min, and detecting the fluorescence value by using a fluorescence enzyme label reader under the excitation wavelength of 365nm and the emission wavelength of 625 nm;
optionally, when the particle size of the CdTe quantum dots is 8nm, diluting the original coating concentration by using a coating buffer solution to be 2 mug/mL, and diluting the CdTe quantum dot marked olaquindox antigen solution by 4 volume times;
optionally, when the particle size of the CdTe quantum dots is 11nm, diluting the original coating concentration by using a coating buffer solution to be 3 mug/mL, and diluting the CdTe quantum dot marked olaquindox antigen solution by 8 times by volume;
optionally, when the particle size of the CdTe quantum dot is 15nm, the coating concentration is diluted by a coating buffer solution to be 5 mug/mL, and the olaquindox antigen solution of the CdTe quantum dot is diluted by 16 times by volume.
A fluorescence immunoassay kit for detecting olaquindox by utilizing CdTe quantum dots comprises the following independently packaged reagents:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS buffer solution, and has the pH value of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Quindox standard solution with the concentration range of mg/mL;
CdTe quantum dot marked olaquindox antigen solution: a method according to any one of claims 1-2;
sealing liquid: 0.1-2% v/v OVA;
coating buffer solution: 0.01-0.05mol/L PBS (pH7.0-7.8), 0.005-0.03mol/L PBST (pH7.4-7.8), 0.005-0.05mol/L CBS (pH8.0-10.2), or 0.005-0.03mol/L Tris-HCl (pH7.8-9.6).
The method for labeling the olaquindox antigen by the CdTe quantum dots or the application of the fluorescence immunoassay kit for detecting olaquindox by utilizing the CdTe quantum dots in the detection of olaquindox;
optionally, the use of olaquindox in the detection of animal meat or animal urine;
optionally, the use of olaquindox in the detection of pork, beef, mutton, horse meat, rabbit meat, pig urine, cow urine, sheep urine, horse urine or rabbit urine.
The technical scheme of the invention has the following advantages:
1. the invention provides a method for labeling a olaquindox antigen by CdTe quantum dots, which comprises the following steps: adding EDC and NHS into the olaquindox antigen solution, standing, then adding CdTe quantum dots, and standing for reaction; the particle size range of the CdTe quantum dots is 8-15 nm; a large number of groping experiments show that when the particle size of the CdTe quantum dots is in the range of 8-15nm, the olaquindox antigen can cover the surface defects of the CdTe quantum dots, and a fluorescent marker with relatively stable fluorescence performance can be formed. When the particle size of the CdTe quantum dots is less than 8nm, olaquindox antigen is excessively adsorbed on the surfaces of the CdTe quantum dots to form new surface defects, and the fluorescent marker is unstable; when the particle size of the CdTe quantum dot is larger than 15nm, the olaquindox antigen can not completely cover the surface of the CdTe quantum dot, and the marker is placed for 24 hours, obvious yellow precipitate appears, which is very unfavorable for establishing an indirect competition fluorescence immunoassay method.
2. The fluorescence immunoassay method for detecting the olaquindox by using the CdTe quantum dots, provided by the invention, has the advantages that the pretreatment is simple, precise large-scale precise instruments and equipment are not needed, particularly, compared with a colloidal gold immunochromatography method, the sensitivity is high, and false positive is solved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is the relationship between the fluorescence intensity of CdTe quantum dot labeled olaquindox antigen solution and the dilution ratio of the reagent in the experimental example 1;
FIG. 2 is a diagram showing the condition optimization of indirect competitive FIA analysis method in Experimental example 1 of the present invention;
FIG. 3 is a standard curve in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The instruments and reagents referred to in the following examples: multifunctional microplate reader (SpectraMax i3x, MD corp.); fluorescence spectrophotometer model F-7000 (Hitachi Co.); u3900 ultraviolet-visible spectrophotometer (hitachi); pHS-3C acidimeter (Shanghai Lei magnetic Co.); GZX-9240MBE electric heating constant temperature drying oven (Shanghai Boxun industries, Ltd.); 79-1 heating magnetic stirrers (Shanghai Pudong physical optical instruments factory); tellurium powder (Te99.999%, Shanghai chemical reagents, China pharmaceutical); cadmium chloride (analytically pure, Shanghai chemical reagents, China pharmaceutical); sodium borohydride (96%, Shanghai chemical reagents, China pharmaceutical); thioglycolic acid (analytical grade, Shanghai chemical reagents, China pharmaceutical); sodium hydroxide (analytical grade, Shanghai chemical reagents, China pharmaceutical); rhodamine 6G (analytical grade, alatin reagent); the experimental water was made by a Milli-Q water treatment system (Millipore). Quinalcohol standards (sigma company), olaquindox antibodies (5mg/mL), and olaquindox antigens (6.2mg/mL) were purchased from Beijing Vierweikang scientific and technological Co. EDC is known by its name 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, NHS is known by its name N-hydroxysulfosuccinimide and OVA is known by its name ovalbumin.
The preparation of the CdTe quantum dot refers to the preparation and characterization of the microwave synthesized water-soluble CdTe quantum dot, wherein the quality of agricultural products is safe, 2015, 2, 51-55, Zhang jin Yan, Wei Yi Hua, and the like.
The sample to be detected in the following examples is prepared by taking 50mL of a pig urine sample or 50g of a pork sample, dissolving the sample in a NaOH solution (1mol/L), filtering and centrifuging (high-speed centrifugation for 3min, wherein the rotation speed is set to 20000 revolutions/min), and taking the supernatant for detection.
Example 1 method for labeling olaquindox antigen by CdTe quantum dot
Taking 0.2mL of olaquindox antigen solution with the concentration of 0.5mg/mL, adding 50 mu L of EDC solution with the concentration of 0.10mol/L and 50 mu L of NHS solution with the concentration of 0.1mol/L, standing at the temperature of 10 ℃ for 15min, then adding 0.1mL of CdTe quantum dot solution with the concentration of 0.001mol/L (the particle size of the quantum dot is 10nm), adding a buffer solution to a constant volume of 2mL, and then standing at the temperature of 4 ℃ for 36 h.
Example 2 method for labeling olaquindox antigen by CdTe quantum dot
Taking 0.2mL of olaquindox antigen solution with the concentration of 0.3mg/mL, adding 50 mu L of EDC solution with the concentration of 0.10mol/L and 50 mu L of NHS solution with the concentration of 0.1mol/L, standing at the temperature of 10 ℃ for 8min, then adding 0.15mL of CdTe quantum dot solution with the concentration of 0.005mol/L (the particle size of the quantum dot is 8nm), adding a buffer solution to a constant volume of 5mL, and then standing at the temperature of 4 ℃ for 48 h.
Example 3 method for labeling olaquindox antigen by CdTe quantum dot
0.3mL of olaquindox antigen solution with the concentration of 0.2mg/mL is taken, 40 mul of EDC solution with the concentration of 0.20mol/L and 40 mul of NHS solution with the concentration of 0.2mol/L are added, the mixture is kept stand for 15min at the temperature of 20 ℃, then 0.20mL of CdTe quantum dot solution with the concentration of 0.004mol/L (the particle size of the quantum dot is 11nm) is added, a buffer solution is added to the mixture to reach the constant volume of 8mL, and the mixture is kept stand for 48h at the temperature of 4 ℃.
Example 4 method for labeling olaquindox antigen by CdTe quantum dot
Taking 0.2mL of olaquindox antigen solution with the concentration of 0.45mg/mL, adding 50 mu L of EDC solution with the concentration of 0.10mol/L and 50 mu L of NHS solution with the concentration of 0.1mol/L, standing at the temperature of 18 ℃ for 15min, then adding 0.15mL of CdTe quantum dot solution with the concentration of 0.005mol/L (the particle size of the quantum dot is 15nm), adding a buffer solution to a constant volume of 10mL, and then standing at the temperature of 4 ℃ for 48 h.
Example 5 fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots
The fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots comprises:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS buffer solution, and has the pH value of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Quindox standard solution within the concentration range of mg/mL;
CdTe quantum dot marked olaquindox antigen solution: marked by the method described in example 1;
sealing liquid: 0.1% v/v OVA;
coating buffer solution: 0.01mol/L PBS pH 7.4.
Example 6 fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots
The fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots comprises:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl, 0.5% v/v PBS buffer solution, and has pH of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Quindox standard solution within the concentration range of mg/mL;
CdTe quantum dot labeled olaquindox antigen solution: marked by the method described in example 2;
sealing liquid: OVA at 0.5% v/v;
coating buffer solution: 0.0lmol/L PBST of pH 7.4.
Example 7 fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots
The fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots comprises:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS buffer solution, and has the pH value of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Quindox standard solution within the concentration range of mg/mL;
CdTe quantum dot marked olaquindox antigen solution: marked by the method described in example 3;
sealing liquid: 1% v/v OVA;
coating buffer solution: 0.01mol/L Tris-HCl pH 8.0.
Example 8 fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots
The fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots comprises:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS buffer solution, and has the pH value of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Quindox standard solution within the concentration range of mg/mL;
CdTe quantum dot marked olaquindox antigen solution: marked by the method described in example 4;
sealing liquid: 2% v/v OVA;
coating buffer solution: 0.05mol/L of CBS, pH 9.6.
Example 9 fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots
The present embodiment provides a fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots, which includes the following steps:
(1) coating:
diluting the olaquindox antibody with a coating buffer solution until the concentration of the olaquindox antibody is 4 mug/mL to obtain a coating solution, then adding 20 microliters of the coating solution into each hole of the ELISA plate, coating overnight (10 hours) at 4 ℃, and washing for 3 times by using a PBS solution with the pH value of 7.4;
(2) and (3) sealing:
adding a sealing solution into the coated enzyme label plate according to the volume of 20 microliter/hole, wherein the sealing condition is that the temperature is 10 ℃, the time is 5 hours, and washing is carried out for 3 times by using a PBS (phosphate buffer solution) with the pH value of 7.4;
(3) drawing a standard curve:
adding a series of olaquindox standard solutions (10) with standard concentration into the enzyme label plate after being sealed -6 、10 -5 、10 -4 、10 -3 、10 -2 、10 -1 mg/mL)20 microliter/hole and CdTe quantum dot-labeled olaquindox antigen solution diluent (diluted 8 volume times by adopting the first diluent) 20 microliter/hole, meanwhile, setting a control solution without olaquindox, adding double distilled water 20 microliter/hole and the CdTe quantum dot-labeled olaquindox antigen solution diluent 20 microliter/hole into the sealed enzyme label plate, then adjusting the pH value of the obtained reaction system to 7.4 by using 0.1mol/L HCl or 0.1mol/L sodium hydroxide, reacting at 37 ℃ for 150min, washing for 3 times by using PBS (phosphate buffer solution) with pH value of 7.4, then detecting the fluorescence value by using a fluorescence microplate reader at an excitation wavelength of 365nm and an emission wavelength of 625nm, taking the lg value of the concentration of the olaquindox standard solution as a horizontal coordinate, and taking the binding rate thereof as a vertical coordinate to draw a standard curve; the binding rate is multiplied by 100 percent of the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox;
(4) measurement of
Adding a sample to be detected (the volume of the added sample is the same as the volume of the added olaquindox standard solution with the series standard concentration in the standard curve drawn in the step (3)) and a CdTe quantum dot marked olaquindox antigen solution diluent (the dilution times and the added volume are the same as those in the standard curve drawn in the step (3)), then the pH value of the obtained reaction system is adjusted to 7.4 by 0.1mol/L HCl or 0.1mol/L NaOH, the reaction is carried out for 150min at 37 ℃, PBS solution with pH value of 7.4 is washed for 3 times, then, the fluorescence value is detected by a fluorescence microplate reader under the excitation wavelength of 365nm and the emission wavelength of 625nm, and (3) calculating the binding rate of the sample to be detected according to the binding rate formula (the binding rate is the fluorescence value of the sample to be detected/the fluorescence value of the control solution without olaquindox multiplied by 100%), and substituting the calculated binding rate into the standard curve to calculate the concentration of the sample to be detected.
Example 10 fluorescence immunoassay method for detecting olaquindox using CdTe Quantum dot
The present embodiment provides a fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots, which includes the following steps:
(1) coating:
diluting the olaquindox antibody with a coating buffer solution until the concentration of the olaquindox antibody is 2 mug/mL to obtain a coating solution, then adding 50 microliters of the coating solution into each hole of the ELISA plate, coating for 12 hours at 4 ℃, and washing for 3 times by using PBS (phosphate buffer solution) with pH7.4;
(2) and (3) sealing:
adding a sealing solution into the coated enzyme label plate according to 50 microliter/hole, and washing for 3 times by using a PBS (phosphate buffer solution) with the pH value of 7.4 under the sealing condition that the temperature is 20 ℃ and the time is 15 hours;
(3) drawing a standard curve:
adding a series of olaquindox standard solutions (10) with standard concentration into the enzyme label plate after being sealed -6 、10 -5 、10 -4 、10 -3 、10 -2 、10 -1 mg/mL)50 microliter/hole and 50 microliter/hole of CdTe quantum dot labeled olaquindox antigen solution diluent (diluted by 4 volume times by adopting the first diluent), meanwhile, setting a control solution without adding olaquindox, adding 50 microliter/hole of double distilled water and 50 microliter/hole of the CdTe quantum dot labeled olaquindox antigen solution diluent into a sealed enzyme label plate, then adjusting the pH value of the obtained reaction system to 7.4 by using 0.1mol/L HCl or 0.1mol/L NaOH, reacting at 37 ℃ for 150min, washing for 3 times by using PBS (phosphate buffer solution) with pH7.4, then detecting the fluorescence values by using a fluorescence microplate reader at an excitation wavelength of 365nm and an emission wavelength of 625nm, taking the lg value of the concentration of the olaquindox standard solution as a horizontal coordinate, and taking the binding rate as a vertical coordinate, and drawing a standard curve; the binding rate is the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox is multiplied by 100 percent;
(4) measurement of
Adding a sample to be detected (the volume of the added sample is the same as the volume of the added olaquindox standard solution with the series standard concentration in the standard curve drawn in the step (3)) and a CdTe quantum dot marked olaquindox antigen solution diluent (the dilution times and the added volume are the same as those in the standard curve drawn in the step (3)), then the pH value of the obtained reaction system is adjusted to 7.4 by 0.1mol/L HCl or 0.1mol/L NaOH, the reaction is carried out for 150min at 37 ℃, PBS solution with pH value of 7.4 is washed for 3 times, then, the fluorescence value is detected by a fluorescence microplate reader under the excitation wavelength of 365nm and the emission wavelength of 625nm, and calculating the binding rate of the sample to be detected according to the binding rate formula (the binding rate is the fluorescence value of the sample to be detected/the fluorescence value of the control solution without olaquindox multiplied by 100%), and substituting the binding rate of the sample to be detected into the standard curve to calculate the concentration of the sample to be detected.
Example 11 fluorescence immunoassay method for detecting olaquindox using CdTe Quantum dot
This example provides a fluorescence immunoassay method for detecting olaquindox using CdTe quantum dots using the kit of example 7, including the following steps:
(1) coating:
diluting the olaquindox antibody with a coating buffer solution until the concentration of the olaquindox antibody is 3 mug/mL to obtain a coating solution, then adding 50 microliters of the coating solution into each hole of the ELISA plate, coating for 16 hours at 2 ℃, and washing for 2 times by using a PBS (phosphate buffer solution) with the pH value of 7.4;
(2) and (3) sealing:
adding 50 microliter of sealing liquid into the coated enzyme label plate, and washing for 3 times by PBS (phosphate buffer solution) with the pH value of 7.4 under the sealing condition of 15 ℃ for 10 hours;
(3) drawing a standard curve:
adding a series of olaquindox standard solutions (10) with standard concentration into the enzyme label plate after being sealed -6 、10 -5 、10 -4 、10 -3 、10 -2 、10 -1 mg/mL)50 microliter/hole and CdTe quantum dot mark olaquindox antigen solution diluent (diluted 8 times by volume by adopting the first diluent) 50 microliter/hole, meanwhile, setting a contrast solution without olaquindox, adding 50 microliter/hole of double distilled water and 50 microliter/hole of CdTe quantum dot mark olaquindox antigen solution diluent into the sealed enzyme label plate, and then obtaining a reaction systemAdjusting the pH value to 7.4 by using 0.1mol/L HCl or 0.1mol/L NaOH, reacting for 150min at 37 ℃, washing for 3 times by using PBS (phosphate buffer solution) with the pH value of 7.4, detecting the fluorescence value by using a fluorescence microplate reader under the excitation wavelength of 365nm and the emission wavelength of 625nm, and drawing a standard curve by using the lg value of the concentration of the olaquindox standard solution as a horizontal coordinate and the binding rate as a vertical coordinate; the binding rate is multiplied by 100 percent of the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox;
(4) measurement of
Adding a sample to be detected (the volume of the added sample is the same as the volume of the olaquindox standard solution with the series standard concentration in the standard curve drawn in the step (3)) and the olaquindox antigen solution diluent marked by the CdTe quantum dots (the dilution times and the added volume are the same as those in the standard curve drawn in the step (3)), then the pH value of the obtained reaction system is adjusted to 7.4 by 0.1mol/L HCl or 0.1mol/L NaOH, the reaction is carried out for 150min at 37 ℃, PBS solution with pH value of 7.4 is washed for 3 times, then, the fluorescence value is detected by a fluorescence microplate reader under the excitation wavelength of 365nm and the emission wavelength of 625nm, and calculating the binding rate of the sample to be detected according to the binding rate formula (the binding rate is the fluorescence value of the sample to be detected/the fluorescence value of the control solution without olaquindox multiplied by 100%), and substituting the binding rate of the sample to be detected into the standard curve to calculate the concentration of the sample to be detected.
Example 12 fluorescence immunoassay method for detecting olaquindox using CdTe Quantum dot
The present embodiment provides a fluorescence immunoassay method for detecting olaquindox by using a CdTe quantum dot, which includes the following steps:
(1) coating:
diluting the olaquindox antibody with a coating buffer solution until the concentration of the olaquindox antibody is 5 mug/mL to obtain a coating solution, then adding 50 microliters of the coating solution into each hole of the ELISA plate, coating for 5 hours at 10 ℃, and washing for 1 time by using a PBS (phosphate buffer solution) with the pH value of 7.4;
(2) and (3) sealing:
adding 50 microliter of sealing liquid into the coated enzyme label plate, and washing for 3 times by PBS (phosphate buffer solution) with the pH value of 7.4 under the sealing condition of 15 ℃ for 10 hours;
(3) drawing a standard curve:
adding a series of olaquindox standard solutions (10) with standard concentration into the enzyme label plate after being sealed -6 、10 -5 、10 -4 、10 -3 、10 -2 、10 -1 mg/mL)50 microliter/hole and 50 microliter/hole of CdTe quantum dot labeled olaquindox antigen solution diluent (diluted by 16 volume times by adopting the first diluent), meanwhile, setting a control solution without adding olaquindox, adding 50 microliter/hole of double distilled water and 50 microliter/hole of the CdTe quantum dot labeled olaquindox antigen solution diluent into a sealed enzyme label plate, then adjusting the pH value of the obtained reaction system to 7.4 by using 0.1mol/L HCl or 0.1mol/L NaOH, reacting at 37 ℃ for 150min, washing for 3 times by using PBS (phosphate buffer solution) with pH7.4, then detecting the fluorescence values by using a fluorescence microplate reader at an excitation wavelength of 365nm and an emission wavelength of 625nm, taking the lg value of the concentration of the olaquindox standard solution as a horizontal coordinate, and taking the binding rate as a vertical coordinate, and drawing a standard curve; the binding rate is multiplied by 100 percent of the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox;
(4) measurement of
Adding a sample to be detected (the volume of the added sample is the same as the volume of the olaquindox standard solution with the series standard concentration in the standard curve drawn in the step (3)) and the olaquindox antigen solution diluent marked by the CdTe quantum dots (the dilution times and the added volume are the same as those in the standard curve drawn in the step (3)), then the pH value of the obtained reaction system is adjusted to 7.4 by 0.1mol/L HCl or 0.1mol/L NaOH, the reaction is carried out for 150min at 37 ℃, PBS solution with pH value of 7.4 is washed for 3 times, then, the fluorescence value is detected by a fluorescence microplate reader under the excitation wavelength of 365nm and the emission wavelength of 625nm, and calculating the binding rate of the sample to be detected according to the binding rate formula (the binding rate is the fluorescence value of the sample to be detected/the fluorescence value of the control solution without olaquindox multiplied by 100%), and substituting the binding rate of the sample to be detected into the standard curve to calculate the concentration of the sample to be detected.
Experimental example 1
1. Performance identification of quantum dot labeled antigen
The labeled antigen is generally characterized by its F/P ratio. The F/P ratio represents the combination ratio of CdTe quantum dot and olaquindox antigen. The procedure of example 1 was followed, the concentration and volume of the raw materials (including the particle size of CdTe quantum dots) were selected as in table 1 below, and the prepared CdTe quantum dot-labeled olaquindox antigen was measured for fluorescence values of 280nm (specific absorption peak of protein) and 450nm (specific absorption peak of labeled CdTe quantum dot) using an ultraviolet spectrophotometer, respectively. F/P value is between 1 and 5, so that a fluorescence immunoassay method is conveniently established. Selecting the optimal complex as clear and transparent yellow liquid, and no precipitation turbidity, stable F/P value, cold storage at 4 deg.C for three weeks, and no great change in fluorescence intensity value and maximum emission peak position, gradually diluting CdTe quantum dot labeled olaquindox antigen solution (PBS of pH7.4), measuring its fluorescence intensity, and making it have good linear relation with the concentration of the conjugate, as shown in FIG. 1, the linear equation is I2681.2C +35.178, and the linear range is 1: 2.5-1: 96 correlation coefficient R 2 0.9978, further illustrates that the prepared CdTe quantum dot labeled olaquindox antigen solution reagent has good dispersibility and stability, and can meet the application of CdTe serving as a fluorescent probe for fluorescence immunoassay. The F/P value is calculated as follows.
The experimental results are as follows:
a great deal of grope is carried out in the experiment, and the experiment finds that the using amounts of CdTe quantum dots with different proportions, olaquindox antigen, EDC and NHS are different, the olaquindox antigen is coated on the surfaces of the CdTe quantum dots with different grain diameters, the F/P value is changed regularly,
when the particle size of the CdTe quantum dots is 8nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.3mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 mu L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 mu L, which is the optimal proportion;
when the particle size of the CdTe quantum dots is 11nm, the concentration of a CdTe quantum dot solution is 0.004mol/L, the added volume is 0.20mL, the concentration of a olaquindox antigen solution is 0.20mg/mL, the added volume is 0.3mL, the concentration of an EDC solution is 0.2mol/L, the added volume is 40 mu L, the concentration of an NHS solution is 0.2mol/L, and the added volume is 40 mu L, which is the optimal proportion;
when the particle size of the CdTe quantum dots is 15nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.45mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 mu L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 mu L, which is the optimal mixture ratio.
The specific results are as follows:
TABLE 1
2. And selecting the optimal working concentration of the CdTe quantum dot labeled antigen and the olaquindox antibody.
The optimum working concentration for indirect competitive fluoroimmunoassay is selected as the CdTe quantum dot-labeled olaquindox antigen and olaquindox antibody concentration for obtaining larger fluorescence value, and the method is implemented according to example 10, except that CdTe quantum dot-labeled antigen (CdTe-ola) (including selection of CdTe quantum dot particle size and dilution) and olaquindox antibody concentration in the following tables 2-4 are adopted, and the detected fluorescence value is shown in the following tables 2-4.
As a result, it was found that:
the optimal working concentration of the CdTe quantum dot labeled antigen and the olaquindox antibody is selected as follows:
when the CdTe particle size is 8nm, the optimal working concentration of the olaquindox antibody is 2 mug/mL, and the optimal dilution of the CdTe quantum dot marked olaquindox antigen solution is 1: 4 (dilution 4 vol); when the CdTe particle size is 11nm, the optimal working concentration of the olaquindox antibody is 3 mug/mL, and the optimal dilution of the CdTe quantum dot marked olaquindox antigen solution is 1: 8 (8-fold dilution by volume); when the CdTe particle size is 15nm, the optimal working concentration of the olaquindox antibody is 5 mug/mL, and the optimal dilution of the CdTe quantum dot marked olaquindox antigen solution is 1: 16 (dilution 16 vol).
TABLE 2 selection of optimal working concentrations of Quinoethanol antibody and labeled antigen (8nm)
TABLE 3 selection of optimal working concentrations of Quinoethanol antibody and labeled antigen (11nm)
TABLE 4 selection of optimal working concentrations of Quinoethanol antibody and labeled antigen (15nm)
2. Influence of the coating Medium
The procedure is as in example 10, except that the coating source is diluted with 0.05mol/L of CBS, pH9.6, 0.01mol/L of PBS, pH7.4, 0.0lmol/L of PBST, pH7.4 and 0.01mol/L of Tris-HCl, pH8.0, respectively.
As a result of experiments, it was found that 0.01mol/L Tris-HCl pH8.0 as a coating buffer gave a high fluorescence intensity and at the same time the best linear relationship of the groups. 0.01mol/L PBS (pH7.4), 0.0lmol/L PBST (pH7.4), and 0.05mol/L CBS (pH9.6) dilute coating effect are respectively given next time.
3. Envelope time optimization
The procedures (1) to (3) in example 10 were carried out, except that the coating time was 5h, 8h, 10h, 12h, and 16h for the olaquindox antibody at 4 ℃.
The experimental results are shown in FIG. 2 (the fluorescence intensity results from left to right in the figure correspond to 5h, 8h, 10h, 12h and 16h in sequence), and the fluorescence intensity gradually increases from 5h to 12h and then becomes stable. The coating time can reach the maximum at 12h, so for the convenience of arranging the experiment, the invention selects 4 ℃ for coating overnight.
4. Optimization of reaction time
The procedures (1) to (3) in example 10 were followed except that the reaction time was 37 ℃ for 30, 60, 90, 150, 180 min.
The experimental results are shown in fig. 2 (the fluorescence intensity results from left to right in the figure correspond to 30, 60, 90, 150 and 180min in sequence), the reaction is maximum at the beginning of the reaction for 150min, and then the reaction is slow, and the fluorescence intensity tends to be stable. After 150min, the reaction time is prolonged, and the fluorescence intensity of the combined compound is not changed greatly. Therefore, the time of the immune response is 150 min.
5. Optimization of pH
The pH of the solution affected the molecular structure of the substance to be measured and also the antibody or antigen, and thus the sensitivity of the reaction, so this experiment was performed as in steps (1) to (3) of example 10, except that the pH was adjusted to 6.5, 7.0, and 7.8 with 0.1mol/L HCl or 0.1mol/L NaOH, respectively, in the pH adjustment step.
The results are shown in fig. 2 (the fluorescence intensity results from left to right in the figure correspond to 6.5, 7.0 and 7.8 in sequence), the fluorescence intensity difference of the system in neutral and weak alkaline environments is large, the fluorescence intensity difference is small when the fluorescence intensity difference is smaller than 6.5 or larger than 8.0, and the pH value is 7.4 because the acidity is controlled by the commonly used PBS solution, the pH value is 7.4.
6. Optimization of ionic strength in the first dilution
The procedures (1) to (3) in example 10 were followed, except that a standard solution of olaquindox and a solution of CdTe quantum dot-labeled olaquindox antigen were diluted with 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0mol/L NaCl in PBS (0.5% by volume) buffer (ph7.4), respectively.
The results are shown in FIG. 2 (the fluorescence intensity results from left to right in the figure correspond to 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0mol/L in order), and the difference in fluorescence intensity of the system tends to increase with the increase in ion intensity, but the increase to a certain level decreases. Therefore, the present invention selects PBS solution containing 0.8mol/L NaCl at pH7.4 to control the ionic strength of the system.
7. Optimization of surfactant in first dilution
The procedures (1) to (3) in example 10 were followed, except that the standard solutions of olaquindox and CdTe quantum dot-labeled olaquindox antigen solutions were diluted with 0.01% v/v, 0.02% v/v, 0.05% v/v, 0.1% v/v, 0.5% v/v, 1% v/vTween-20 (0.5% by volume) PBS buffer (pH7.4), respectively.
The results are shown in FIG. 2 (the fluorescence intensity results from left to right in the figure correspond to 0.01% v/v, 0.02% v/v, 0.05% v/v, 0.1% v/v, 0.5% v/v, and 1% v/v in this order), the influence of Tween-20 on the fluorescence intensity of the system is small, and the concentration of Tween-20 is selected to be 0.1% v/v which is a common concentration.
8. Optimization of confining liquids
The procedures from step (1) to step (3) in example 10 were followed, except that the blocking solutions were 0.1%, 0.5%, 1% and 2% OVA,
The results are shown in FIG. 2 (the fluorescence intensity results from left to right in the figure correspond to 0.1%, 0.5%, 1% and 2% OVA in order), and the fluorescence intensity of the 0.5% OVA-containing solution is the strongest and more stable.
Experimental example 2
1. Drawing of standard curve and determination of actual sample
According to examples 10, 11 and 12, in 10 -6 –10 -1 Examining the concentration range of mg/mL, carrying out 11 parallel measurements for each concentration, taking an average value, drawing a standard curve by taking the lg value of the olaquindox concentration as an abscissa and the binding rate as an ordinate, and as shown in FIG. 3, sequentially setting the linear regression equations as follows:
the grain diameter of CdTe quantum dot is 8nm, I is 8.6286lgC +63.2(I is binding rate, C is concentration of olaquindox), and the correlation coefficient is R 2 0.9841 detection limit IC 20 ＝9.86×10 -6 mg/mL，IC 50 ＝2.9×10 -3 mg/mL。
The grain diameter of CdTe quantum dot is 11nm, I is 8.1143lgC +64.067(I is binding rate, C is concentration of olaquindox), and the correlation coefficient is R 2 0.9888 detection limit IC 20 ＝3.71×10 -6 mg/mL，IC 50 ＝6.33×10 -5 mg/mL。
The grain diameter of CdTe quantum dot is 15nm, I is 5.714lgC +63.33(I is binding rate, C is concentration of olaquindox), and the correlation coefficient is R 2 0.9796 detection limit IC 20 ＝2.63×10 -8 mg/mL，IC 50 ＝4.68×10 -3 mg/mL。
The results of the fluorescence immunoassay and spiking recovery experiments performed on the samples tested are shown in table 5:
TABLE 5 antigen-coated fluoroimmunoassay recovery experiment of olaquindox in livestock and poultry products
As can be seen from the table, when the added concentration is lower, the obtained recovery rate is 92.2-123%, and the relative standard deviation is less than or equal to 10%, which shows that the recovery effect is good, the test method is reliable, the result is satisfactory, and the requirement of detecting trace olaquindox in livestock and poultry products can be met.
2. Precision of the method
The coefficient of variation of olaquindox detected by the antibody-coated fluoroimmunoassay method performed according to example 10 (the olaquindox concentration in the test sample is shown in table 6) is shown in table 6 by repeated experiments within and between batches. It is understood that the coefficient of variation between batches is relatively high, probably due to differences in binding resulting from the reaction of the antibody antigen to the manipulation during the day, whereas the intra-day differences are relatively small, so that the intra-batch errors are relatively low.
TABLE 6 precision test results of antigen-coated fluoroimmunoassay for olaquindox detection
3 specificity of the method
Selecting salbutamol, clenbuterol hydrochloride,The specificity of the method was evaluated by performing an antibody cross-reaction experiment with ractopamine, mabuterol, bambuterol, and brombuterol (diluting other analogs with similar chemical structures to a series of concentrations, substituting for standard solutions of the substances to be tested, and determining the respective cross-reaction rates by the experimental method 50 FL/IC 50 . Other structural analogs) × 100%, IC 50 Concentration of the analog to be detected, IC, corresponding to 50% of the maximum quenched fluorescence intensity 50 FL represents the concentration of olaquindox at 50% of the maximum quenched fluorescence intensity, IC 50 . Other structural analogs are expressed as the concentration of the corresponding other structural analog at 50% of the maximum quenched fluorescence intensity. ) When the binding rate of the olaquindox to the antigen antibody response is 50%, the cross-reactivity of olaquindox to other beta-receptor agonists is low, and the effect is not considered to be influenced.
TABLE 7 Cross-reactivity of beta-receptor agonists
The invention inspects a plurality of factors in the fluorescence immunoassay, and the influence of the optimum concentration, the coating medium, the coating pH value, the ionic strength, the surfactant and the confining liquid on the fluorescence strength of the system, optimizes the system conditions and develops the research of detecting the olaquindox by the indirect competition fluorescence immunoassay method.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (9)
1. A method for labeling a olaquindox antigen by CdTe quantum dots is characterized by comprising the following steps: adding EDC solution and NHS solution into the olaquindox antigen solution, standing, then adding CdTe quantum dots, and standing for reaction; the particle size range of the CdTe quantum dots is 8-15 nm.
2. The method for labeling the olaquindox antigen by the CdTe quantum dot as claimed in claim 1, wherein 0.1-0.5mL of olaquindox antigen solution with the concentration of 0.1-0.8mg/mL is taken, 20-60 μ L of EDC solution with the concentration of 0.05-0.30mol/L and 20-60 μ L of NHS solution with the concentration of 0.05-0.30mol/L are added, the mixture is allowed to stand at 10-26 ℃ for 8-20min, then 0.05-0.5mL of CdTe quantum dot solution with the concentration of 0.001-0.005mol/L is added, buffer solution is added to the mixture to reach 2-10mL, and then the mixture is allowed to stand at 0-6 ℃ for 36-48 h;
optionally, 0.2mL of olaquindox antigen solution with the concentration of 0.5mg/mL is taken, 50 muL of EDC solution with the concentration of 0.10mol/L and 50 muL of NHS solution with the concentration of 0.1mol/L are added, standing is carried out for 15min at the temperature of 10-26 ℃, then 0.1mL of CdTe quantum dot solution with the concentration of 0.001mol/L is added, buffer solution is added to the solution to reach the constant volume of 5mL, and then the solution is placed for 36-48h at the temperature of 0-6 ℃;
optionally, when the particle size of the CdTe quantum dots is 8nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.3mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 μ L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 μ L;
optionally, when the particle size of the CdTe quantum dot is 11nm, the concentration of the CdTe quantum dot solution is 0.004mol/L, the added volume is 0.20mL, the concentration of the olaquindox antigen solution is 0.20mg/mL, the added volume is 0.3mL, the concentration of the EDC solution is 0.2mol/L, the added volume is 40 μ L, the concentration of the NHS solution is 0.2mol/L, and the added volume is 40 μ L;
optionally, when the particle size of the CdTe quantum dot is 15nm, the concentration of the CdTe quantum dot solution is 0.005mol/L, the added volume is 0.15mL, the concentration of the olaquindox antigen solution is 0.45mg/mL, the added volume is 0.2mL, the concentration of the EDC solution is 0.1mol/L, the added volume is 50 μ L, the concentration of the NHS solution is 0.1mol/L, and the added volume is 50 μ L.
3. The use of the CdTe quantum dot-labeled olaquindox antigen marked by the method of any one of claims 1-2 in detecting olaquindox;
optionally, the use of olaquindox in the detection of animal meat or animal urine;
optionally, use in detecting pork, beef, mutton, horse meat, rabbit meat, pig urine, cow urine, sheep urine, horse urine or rabbit urine.
4. A fluorescence immunoassay method for detecting olaquindox by utilizing CdTe quantum dots is characterized by comprising the following steps:
coating: using a olaquindox antibody as a coating antigen, diluting the coating antigen with a coating buffer solution to obtain a coating solution, coating an ELISA plate, and washing;
and (3) sealing: sealing the coated ELISA plate with a sealing liquid, and washing;
drawing a standard curve: adding a series of olaquindox standard solutions with standard concentrations and a CdTe quantum dot labeled olaquindox antigen solution diluent into the closed enzyme label plate to perform indirect competitive reaction, simultaneously setting a control solution without olaquindox, washing the plate, detecting the fluorescence value, taking the lg value of the olaquindox standard solution concentration as a horizontal coordinate, taking the binding rate as a vertical coordinate, and drawing a standard curve; the CdTe quantum dot marked olaquindox antigen solution is marked by the method of any one of claims 1-2; the binding rate is multiplied by 100 percent of the fluorescence value of the olaquindox standard solution/the fluorescence value of the control solution without olaquindox;
and (3) determination: and adding a sample to be detected and a CdTe quantum dot-labeled olaquindox antigen solution diluent into the closed enzyme label plate to perform indirect competitive reaction, washing the plate, detecting the fluorescence value, calculating the binding rate, and substituting the obtained binding rate into the standard curve to calculate the concentration of the sample to be detected.
5. The fluoroimmunoassay method for detecting olaquindox using CdTe quantum dots of claim 4, wherein the coating buffer is 0.01-0.05mol/L PBS (pH 7.0-7.8), 0.005-0.03mol/L PBST (pH 7.4-7.8), 0.005-0.05mol/L CBS (pH 8.0-10.2) or 0.005-0.03mol/L Tris-HCl (pH 7.8-9.6);
optionally, the coating buffer is 0.01mol/L PBS (pH7.4), 0.0lmol/L PBST (pH7.4), 0.05mol/L CBS (pH9.6) or 0.01mol/L Tris-HCl (pH8.0);
optionally, the coating buffer is 0.01mol/L Tris-HCl with pH value of 8.0;
optionally, the olaquindox antibody is used as a coating antigen, the concentration of the coating antigen is diluted to 2-5 mug/mL by using a coating buffer solution to obtain a coating solution, then an ELISA plate is added according to 20-50 microliter/hole, and the coating is carried out for 5-16h at the temperature of 2-10 ℃.
6. The fluorescence immunoassay method for detecting olaquindox by using CdTe quantum dots as claimed in claim 4 or 5, wherein the blocking solution is 0.1-2% v/v ovalbumin;
optionally, the blocking solution is 0.1% v/v, 0.5% v/v, 1% v/v or 2% v/vOVA;
optionally, the confining liquid is 0.5% v/v OVA;
optionally, adding blocking liquid into the coated enzyme label plate according to 20-50 microliters/hole, wherein the blocking condition is that the temperature is 10-20 ℃ and the time is 5-15 hours.
7. The fluoroimmunoassay method for detecting olaquindox using CdTe quantum dots according to any one of claims 4 to 6, wherein in the step of drawing the standard curve, a series of olaquindox standard solutions with standard concentration and a dilution of CdTe quantum dot-labeled olaquindox antigen solution are obtained by diluting with a first dilution, and the concentration range of the series of olaquindox standard solutions with standard concentration is 10 -6 –10 -1 mg/mL, wherein the dilution multiple of the CdTe quantum dot marked olaquindox antigen solution is 4-16 times by volume; the first diluent is a buffer solution containing 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS, and the pH value is 7.4;
optionally, adding 20-50 microliters/hole of a series of olaquindox standard solutions with standard concentrations and 20-50 microliters/hole of a CdTe quantum dot labeled olaquindox antigen solution diluent into the closed enzyme label plate, adjusting the pH value to 7.4, and reacting at 37 ℃ for at least 150 min;
optionally, adding a series of olaquindox standard solutions with standard concentration 50 microliter/pore and olaquindox antigen solution diluent marked by CdTe quantum dots 50 microliter/pore into the sealed enzyme label plate, adjusting the pH value to 7.4, reacting at 37 ℃ for at least 150min, and detecting the fluorescence value by using a fluorescence enzyme label reader under the excitation wavelength of 365nm and the emission wavelength of 625 nm;
optionally, when the particle size of the CdTe quantum dots is 8nm, diluting the original coating concentration by using a coating buffer solution to be 2 mug/mL, and diluting the CdTe quantum dot marked olaquindox antigen solution by 4 volume times;
optionally, when the particle size of the CdTe quantum dots is 11nm, diluting the original coating concentration by using a coating buffer solution to be 3 mug/mL, and diluting the CdTe quantum dot marked olaquindox antigen solution by 8 times by volume;
optionally, when the particle size of the CdTe quantum dot is 15nm, the coating concentration is diluted by a coating buffer solution to be 5 mug/mL, and the olaquindox antigen solution of the CdTe quantum dot is diluted by 16 times by volume.
8. A fluorescence immunoassay kit for detecting olaquindox by utilizing CdTe quantum dots is characterized by comprising the following independently packaged reagents:
first diluent: contains 0.1% v/v Tween-20, 0.8mol/L NaCl and 0.5% v/v PBS buffer solution, and has the pH value of 7.4;
a series of olaquindox standard solutions of standard concentration: diluting olaquindox with a first diluent to obtain 10 -6 –10 -1 Olaquindox standard solution with the concentration range of mg/mL;
CdTe quantum dot marked olaquindox antigen solution: a method according to any one of claims 1-2;
sealing liquid: 0.1-2% v/v OVA;
coating buffer solution: 0.01-0.05mol/L PBS (pH7.0-7.8), 0.005-0.03mol/L PBST (pH7.4-7.8), 0.005-0.05mol/L CBS (pH8.0-10.2), or 0.005-0.03mol/L Tris-HCl (pH7.8-9.6).
9. The method for labeling olaquindox antigen by using CdTe quantum dots as claimed in any one of claims 1 to 2 or the use of the fluorescence immunoassay kit for detecting olaquindox by using CdTe quantum dots as claimed in claim 8 in detecting olaquindox;
optionally, the use of olaquindox in the detection of animal meat or animal urine;
optionally, the use of olaquindox in the detection of pork, beef, mutton, horse meat, rabbit meat, pig urine, cow urine, sheep urine, horse urine or rabbit urine.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027424A1 (en) * | 2002-09-19 | 2004-04-01 | Hamamatsu Photonics K.K. | Fluorescence analysis method with the use of fluorescent antibody |
| CN102928590A (en) * | 2012-11-22 | 2013-02-13 | 上海师范大学 | Kit adopting fluorescent quantum dots to quickly screen, separate and detect salmonella |
| CN103353523A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Phosphorescent silica nanoparticle labeled immunity chromatography method and test paper thereof |
| CN103361064A (en) * | 2013-06-03 | 2013-10-23 | 中国人民解放军第二军医大学 | Preparation method of signal amplifying type quantum dot immune fluorescent probe and application of signal amplifying quantum dot immune fluorescent probe |
| CN106770210A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of olaquindox method for quick |
| CN108152257A (en) * | 2017-12-12 | 2018-06-12 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of method that Zilpaterol is measured using CdTe quantum |
| CN109837083A (en) * | 2017-11-24 | 2019-06-04 | 中国农业大学 | Difunctional fluorescence nano flower of a kind of hybrid and the preparation method and application thereof |
| CN111089960A (en) * | 2020-01-13 | 2020-05-01 | 佛山职业技术学院 | Quinoethanol rapid detection device |
-
2022
- 2022-04-11 CN CN202210374966.6A patent/CN114858767A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027424A1 (en) * | 2002-09-19 | 2004-04-01 | Hamamatsu Photonics K.K. | Fluorescence analysis method with the use of fluorescent antibody |
| CN102928590A (en) * | 2012-11-22 | 2013-02-13 | 上海师范大学 | Kit adopting fluorescent quantum dots to quickly screen, separate and detect salmonella |
| CN103353523A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Phosphorescent silica nanoparticle labeled immunity chromatography method and test paper thereof |
| CN103361064A (en) * | 2013-06-03 | 2013-10-23 | 中国人民解放军第二军医大学 | Preparation method of signal amplifying type quantum dot immune fluorescent probe and application of signal amplifying quantum dot immune fluorescent probe |
| CN106770210A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of olaquindox method for quick |
| CN109837083A (en) * | 2017-11-24 | 2019-06-04 | 中国农业大学 | Difunctional fluorescence nano flower of a kind of hybrid and the preparation method and application thereof |
| CN108152257A (en) * | 2017-12-12 | 2018-06-12 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of method that Zilpaterol is measured using CdTe quantum |
| CN111089960A (en) * | 2020-01-13 | 2020-05-01 | 佛山职业技术学院 | Quinoethanol rapid detection device |
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