CN110927383A - Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof - Google Patents

Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof Download PDF

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CN110927383A
CN110927383A CN201910948223.3A CN201910948223A CN110927383A CN 110927383 A CN110927383 A CN 110927383A CN 201910948223 A CN201910948223 A CN 201910948223A CN 110927383 A CN110927383 A CN 110927383A
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olaquindox
solution
sample
test strip
pad
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王赛赛
金仁耀
翟璐
郭建军
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Hangzhou Baixin Technology Co Ltd
Zhejiang Gongshang University
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Hangzhou Baixin Technology Co Ltd
Zhejiang Gongshang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Abstract

The invention discloses a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residue and application thereof, the test strip can carry out qualitative and quantitative detection on olaquindox in water and feed, the pretreatment process of a sample is simple, the test strip is convenient and quick, the detection time is short, and the test strip has high precision and sensitivity; the anti-olaquindox monoclonal antibody prepared by the invention has strong specificity, and the luminescent intensity and detection signal of the fluorescent microsphere can be enhanced along with the enhancement of the excitation light intensity by adopting the anti-olaquindox monoclonal antibody marked by the fluorescent microsphere, so that the analytical sensitivity of the immunochromatography technology can be effectively improved by marking the anti-olaquindox monoclonal antibody by the fluorescent microsphere, and compared with the traditional colloidal gold immunochromatography, the analytical method has higher sensitivity; meanwhile, the fluorescent microsphere has a relatively stable morphological structure, so that the microsphere is uniform in particle size, good in dispersity and stability, high in luminous efficiency and good in repeatability, and the fluorescence quenching of the dye is greatly reduced.

Description

Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof
Technical Field
The invention belongs to the technical field of time-resolved fluoroimmunoassay in biotechnology, and particularly relates to a fluorescent microsphere immunochromatography test strip for detecting olaquindox residues and application thereof.
Background
Olaquindox (OLA) is an antibacterial growth promoter, which has been widely used in aquaculture and is once referred to as "aquatic clenbuterol". The toxic and side effects of olaquindox are not small and have obvious genetic toxicity and accumulative toxicity, so that strict use specifications and residual limit standards are established at home and abroad. If the use of olaquindox is prohibited in the United states and European Union, the Maximum Residual Limit (MRL) of olaquindox in animal tissues and internal organs is defined in Japan as 300. mu.g.kg-1The addition amount of the feed additive in the feed is not higher than 50 mg/kg according to the No. 168 bulletin feed drug additive use Specification published in 2001 by Ministry of agriculture in China-1Meanwhile, the use of the feed in the culture process of fish, poultry and pigs with the weight of more than 35kg is forbidden. Nevertheless, the phenomenon that olaquindox with good antibacterial and growth promoting effects and low price is illegally added and used still exists. Therefore, it is necessary to enhance the detection supervision of olaquindox, especially to enhance the research of olaquindox detection technology.
The method for detecting the residue of olaquindox mainly comprises two main types of traditional instrumental analysis and immunoassay. The instrument method mainly comprises a spectrum method, a chromatography method, a liquid chromatography-mass spectrometry technology and the like, the instrument analysis accuracy is high, the precision is high, but the sample pretreatment process is complex and tedious, the time consumption is long, the operation of professional technicians is needed, the instrument reagent and the like are expensive, and the instrument method cannot be greatly popularized in the basic level. The immunoassay technology is widely applied to the detection of small molecule drug residues by virtue of the advantages of high efficiency, rapidness, high sensitivity, high specificity and the like. The currently commonly used immunodetection methods mainly comprise enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescence immunoassay and the like, but have the problems of unsatisfactory sensitivity, false positive, false negative and the like.
Disclosure of Invention
In order to solve the existing problems, the invention provides a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues comprises a base plate, a sample combination pad, a nitrocellulose membrane and a water absorption pad; the sample combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the bottom plate, the tail end of the sample combination pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample combination pad is aligned with the initial end of the bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the bottom plate; the sample combination pad is embedded with an anti-olaquindox monoclonal antibody marked by fluorescent microspheres;
a detection area and a quality control area are fixed on the nitrocellulose membrane; the detection area is positioned at one side close to the sample combination pad, the quality control area is positioned at one side far away from the sample combination pad, a goat anti-mouse secondary antibody is sprayed on the quality control area, a olaquindox hapten-carrier protein conjugate is sprayed on the detection area, the olaquindox hapten-carrier protein conjugate is an olanexin hapten-ovalbumin conjugate OLA-A-OVA,
the olaquindox hapten-ovalbumin conjugate is prepared by the following steps:
(1) synthesis of olaquindox hapten
(1.1) dissolving 1mmol of olaquindox in 2-5mL of THF, adding 1.0-1.2mmol of NaH at 0 ℃, stirring for 0.5-2 hours, adding 1mmol of 5-bromo-2, 4-diene ethyl valerate, reacting for 6-8 hours at 65 ℃, and adding 30-50mL of H after the reaction is completed2O, extracting with ethyl acetate, mixing organic phases, washing with saturated saline solution, spin-drying solvent, and separating by column chromatography to obtain OLA-A1，A1is-CH-COOCH2CH3；
(1.2) adding 1mmol of OLA-A1Dissolving in 8-12mL of mixed solution of methanol and water, wherein the volumes of the methanol and the water are both 5mL), adding 1-1.5mmol of lithium hydroxide, stirring, and reacting at room temperature for 1-3 h; after the reaction is completed, 1mol/L hydrochloric acid solution is used for adjusting the pH value to be 5-6, ethyl acetate is used for extraction, an organic phase is washed by saturated saline solution and then dried by anhydrous sodium sulfate, and the solvent is removed by rotary evaporation, so that a product OLA-A is-CH-COOH; the specific synthetic route is as follows:
Figure BDA0002224662780000031
(2) synthesis of olaquindox artificial antigen
Dissolving 0.04mmol of OLA-A in 0.8-1.0mL of DMF, adding 0.04mmol of N-hydroxysuccinimide and 0.04mmol of dicyclohexylcarbodiimide, stirring at room temperature in the dark for 10-12h, centrifuging at 2000r/min for 10min, and taking the supernatant as a solution a;
weighing 20mg of OVA (or BSA) and dissolving in 5mL of 0.01mmol/L phosphate buffer solution with pH 7.4 to obtain solution b;
dropwise adding 0.6mL of the solution a into the solution b at 4 ℃, and stirring and reacting at 4 ℃ overnight; transferring into dialysis bag the next day, dialyzing with 0.01mmol/L phosphate buffer solution with pH of 7.4 for 2 days, centrifuging, removing precipitate to obtain crosslinked product, and naming as OLA-A-OVA or OLA-A-BSA, -A-represents-CH-COO-; the specific synthetic route is as follows:
Figure BDA0002224662780000041
further, the bottom plate is a polyvinyl chloride bottom plate; the sample combining pad is glass wool; the absorbent pad is absorbent paper.
Furthermore, the olaquindox monoclonal antibody is obtained by coupling olaquindox hapten with carrier protein to prepare artificial antigen, immunizing a mouse and screening, wherein the carrier protein is ovalbumin or bovine serum albumin.
Further, the fluorescent microspheres are microspheres with the diameter of 100-300 nm and are prepared by wrapping fluorescent materials with polystyrene, carboxyl functional groups are connected to the surfaces of the microspheres, and the fluorescent materials are complexes of europium. The excitation wavelength of the fluorescent microsphere is 365nm, and the emission wavelength is 610 nm.
Further, the preparation of the fluorescent microsphere labeled anti-olaquindox monoclonal antibody comprises the following steps:
1) and (3) activation: suspending 100 μ L microsphere suspension with embedded fluorescent dye and modified carboxyl functional group on surface in 900 μ L activation buffer solution at 4 deg.C for 10000r min-1Centrifuging for 10min, removing supernatant, resuspending microspheres with 1mL of activation buffer for 2 times, adding 200 μ L of activating agent, mixing, and activating with shaking at room temperature for 15 min. The microsphere is a microsphere with the diameter of 100-300 nm and a fluorescent substance wrapped by polystyrene, the surface of the microsphere is connected with-COOH groups, and the fluorescent substance is a europium complex;
2) coupling: mixing the above mixed solution 10000 r.min-1Centrifuging for 10min, removing supernatant, adding 100-200 μ L olaquindox monoclonal antibody (1 mg. mL)-1) Oscillating and reacting for 2 hours at room temperature;
3) and (3) sealing: after the oscillation reaction, the reaction solution is treated at the temperature of 4 ℃ for 5000r min-1Centrifuging for 15min, removing supernatant, resuspending with blocking buffer solution, and sealing at room temperature for 1 hr;
4) and (3) storage: the reaction solution after the sealing is carried out at 4 ℃ for 12000 r.min-1Centrifuging for 15min, removing supernatant, resuspending with storage buffer solution, centrifuging, discarding supernatant, suspending with storage solution again, and storing at 4 deg.C in dark place;
the pH of the activation buffer is 6.0, and 0.05 mol.L-12- (N-morpholine) ethanesulfonic acid buffer (MES);
the activating agent is water-soluble carbodiimide, wherein the molar mass ratio of EDC to NHS to COOH is (2-4) to (10-20) to 1, and the activating agent is diluted to a required concentration by using an activating buffer solution before use;
the pH value of the coupling buffer solution is 7.5-8.5, and the pH value is 0.05 mol.L-1The borate buffer of (1);
the blocking buffer solution contains 0.1-0.4 mol.L-1Ethanolamine, BSA (bovine serum albumin) with the mass fraction of 2-5% and pH of 7.4Phosphate buffer;
the storage buffer solution contains 0.01 percent of N by mass fractionaN3And 0.5% by mass of BSA, pH 7.4.
Further, the preparation of the sample conjugate pad comprises the following steps:
3) the sample binding pad was washed with 0.5% Bovine Serum Albumin (BSA), pH 7.4, 0.1 mol. L-1Soaking in phosphate buffer solution for 2h, and drying at 37 ℃ for 2 h;
4) diluting the stored anti-olaquindox monoclonal antibody marked by the fluorescent microspheres with a storage buffer solution, soaking the sample bonding pad treated in the step 1) in the diluted solution for 30min, and then drying in vacuum for later use; the storage buffer solution contains 0.01 percent of NaN by mass fraction3And 0.5% by mass of BSA, pH 7.4.
An application of a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues in detection of olaquindox is disclosed, wherein the test strip is the test strip, and the method comprises the following steps:
1) pretreating the sample to obtain a sample to be detected;
2) and (3) detecting by using a test strip: sucking 100 mu L of sample solution to be detected, vertically dripping the sample solution to be detected on a test strip sample wafer bonding pad, starting timing when the liquid flows, reacting for 10min, and inserting the test strip into a fluorescence detector for detection to obtain a detection result;
3) and (4) analyzing results: after the test is finished, the instrument calculates the concentration of olaquindox in the sample according to the intensity of the fluorescence signal obtained by detection through a calculation program set by the system, and gives out positive and negative judgment according to a preset threshold value. Because each batch of test strips can carry out a large number of standard curve tests in the early stage to determine the batch of standard curves, if the batch of test strips is used for detecting actual new samples, and when a detection result is calculated, calculation can be directly carried out aiming at the previously fitted standard curves, and a calculation chip in an instrument system carries out processing to obtain the olaquindox concentration.
The invention has the beneficial effects that:
(1) the test strip can be used for qualitatively and quantitatively detecting the olaquindox in water and feed, the pretreatment process of a sample is simple, convenient and fast, the detection time is short, and the test strip has high precision and sensitivity.
(2) The anti-olaquindox monoclonal antibody prepared by the invention has strong specificity, and the luminescent intensity and detection signal of the fluorescent microsphere can be enhanced along with the enhancement of the excitation light intensity by adopting the anti-olaquindox monoclonal antibody marked by the fluorescent microsphere, so that the analysis sensitivity of the immunochromatography technology can be effectively improved by marking the anti-olaquindox monoclonal antibody by the fluorescent microsphere, and compared with the traditional colloidal gold immunochromatography, the immunoassay method disclosed by the invention has higher sensitivity; meanwhile, the fluorescent microsphere has a relatively stable morphological structure, so that the microsphere is uniform in particle size, good in dispersity and stability, high in luminous efficiency and good in repeatability, and the fluorescence quenching of the dye is greatly reduced.
Drawings
FIG. 1 is a schematic view of a cross-sectional structure of a fluorescent microsphere immunochromatographic test strip.
FIG. 2 is a top view of the fluorescent microsphere immunochromatographic test strip.
FIG. 3 shows the synthetic route of olaquindox hapten.
FIG. 4 is the synthetic route of olaquindox artificial antigen.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention.
The substances and detection instruments used in the following examples were commercially available.
The PBS buffers used in the following examples were, unless otherwise specified, all at pH 7.4 and 0.01 mol. L-1Phosphate buffer of (4); all CBS buffers used in the examples were 0.05mol · L at pH 9.6-1The carbonate buffer of (4); bovine serum albumin is BSA for short; ovalbumin is called OVA for short, keyhole limpet hemocyanin is called KLH for short, and olaquindox is called OLA for short.
The relevant solutions used in the following examples were formulated:
PBST lotion: 500mL of the suspension was taken at pH 7.4 and 0.01 mol. L-1Adding 0.25mL of Tween 20 into the phosphate buffer solution, and uniformly mixing for later use.
Sealing liquid: dissolving 1g skimmed milk powder in 50mL pH 7.4, 0.01 mol.L-1Phosphate buffer.
pH＝9.6，0.05mol·L-1Carbonate Buffer (CBS): weighing Na2CO31.59g，NaHCO32.93g, adding pure water to 990mL, adjusting pH to 9.6, adding pure water to 1000mL, and storing at 4 deg.C for use.
0.01mol·L-1Phosphate Buffered Saline (PBS) at pH 7.4: 8.5g NaCl, 2.2g Na2HPO4·12H2O，0.2g NaH2PO4·2H2And O, dissolving in 900mL of pure water, adjusting the pH value to 7.4, and diluting to 1000 mL.
Composition of time-resolved fluorescent microsphere immunochromatographic test strip for detecting olaquindox residue
As shown in fig. 1, the test strip is composed of a bottom plate 6, a sample combination pad 1, a nitrocellulose membrane 2 and a water absorption pad 3; the sample combination pad 1, the nitrocellulose membrane 2 and the water absorption pad 3 are sequentially stuck on the bottom plate 6, the tail end of the sample combination pad 1 is connected with the initial end of the nitrocellulose membrane 2, the tail end of the nitrocellulose membrane 2 is connected with the initial end of the water absorption pad 3, the initial end of the sample combination pad 1 is aligned with the initial end of the bottom plate 6, and the tail end of the water absorption pad 3 is aligned with the tail end of the bottom plate 6;
a detection area 4 and a quality control area 5 are fixed on the nitrocellulose membrane 2; the detection region 4 is located at a side close to the sample pad 1, and the quality control region 5 is located at a side far from the sample pad 1, both of which are strip-shaped and arranged in parallel and have a length direction perpendicular to the length direction of the strip, as shown in fig. 2. The detection area 4 is sprayed with a olaquindox hapten-carrier protein conjugate, wherein the olaquindox hapten-carrier protein conjugate is an olaquindox hapten-ovalbumin conjugate, and the quality control area 5 is sprayed with a goat anti-mouse secondary antibody.
The bottom plate 6 is a polyvinyl chloride bottom plate (PVC bottom plate); the sample combining pad 1 is glass wool; the absorbent pad 3 is absorbent paper.
Preparation of time-resolved fluorescent microsphere immunochromatography test strip for detecting olaquindox residue
The preparation method of the time-resolved fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues mainly comprises the following steps:
1) preparation of sample conjugate pad 1: using a time-resolved fluorescent microsphere produced by Bangs Laboratories to mark an anti-olaquindox monoclonal antibody, diluting the anti-olaquindox monoclonal antibody by using a buffer system, soaking the sample combined pad 1 in a dilution buffer solution, and preparing the anti-olaquindox monoclonal antibody after vacuum freeze drying;
the fluorescent microspheres are microspheres with the diameter of 100-300 nm and are prepared by wrapping fluorescent materials with polystyrene, the surfaces of the microspheres are connected with-COOH groups, and the fluorescent materials are europium complexes.
2) Preparation of nitrocellulose membrane 2: spraying the olaquindox hapten-carrier protein conjugate to a detection area range on the nitrocellulose membrane 2 to prepare a detection area 4; spraying a goat-anti-mouse secondary antibody to the range of the quality control area on the nitrocellulose membrane 2 to prepare a quality control area 5;
3) assembling and shearing: a sample binding pad 1 embedded with a time-resolved fluorescent microsphere labeled anti-olaquindox monoclonal antibody, a nitrocellulose membrane 2 fixed with a detection zone 4 and a quality control zone 5 and a water absorption pad 3 are sequentially bonded on a bottom plate 6 and cut into required widths, namely the olaquindox fluorescent microsphere immunochromatographic test strip.
Example 1
Synthesis of olaquindox artificial antigen
(1) Synthesis of olaquindox hapten
(1.1) dissolving 1mmol of olaquindox in 2-5mL of THF, adding 1.1-1.2mmol of NaH under ice bath (0 ℃), stirring for 1 hour, adding 1mmol of 5-bromo-2, 4-ethyl dienvalerate (A), refluxing at 65 ℃ for 6 hours, and adding 30mL of H after the reaction is completed2O, extracting with ethyl acetate, mixing organic phases, washing with saturated saline solution, removing solvent by rotary evaporation, and separating by column chromatography to obtain OLA-A1，A1is-CH-COOCH2CH3(ii) a The eluent is: petroleum ether: ethyl acetate 1: 1;
(1.2) mixing1mmol OLA-A1Dissolving in 10mL of mixed solution of methanol and water (the volume of the methanol and the water is 5mL), adding 1.5mmol of lithium hydroxide, and reacting for 2h at room temperature; after the reaction is completed, adjusting the pH value to be 5-6 by using 1mol/L hydrochloric acid solution, extracting twice by using ethyl acetate 50ml, washing an organic phase by using saturated saline solution, drying by using anhydrous sodium sulfate, and removing a solvent by rotary evaporation to obtain a product OLA-A, wherein A is-CH-COOH; the specific synthetic route is shown in FIG. 3.
(2) Synthesis of olaquindox artificial antigen
Dissolving 0.04mmol of OLA-A in 0.8-1.0mL of DMF, adding 0.04mmol of N-hydroxysuccinimide and 0.04mmol of dicyclohexylcarbodiimide, stirring at room temperature in the dark for 10h, centrifuging at 2000r/min for 10min, and taking the supernatant as a liquid a after centrifugation;
weighing 20mg of carrier protein OVA (or BSA) and dissolving in 5mL of 0.01mmol/L phosphate buffer solution with pH 7.4 to obtain solution b;
dropwise adding 0.6mL of the solution a into the solution b at 4 ℃, and stirring and reacting at 4 ℃ overnight; transferring into dialysis bag the next day, dialyzing with 0.01mmol/L phosphate buffer solution with pH of 7.4 for 2 days, centrifuging, removing precipitate to obtain crosslinked product, and naming as OLA-A-OVA or OLA-A-BSA, -A-represents-CH-COO-; the specific synthetic route is shown in FIG. 4; m and n respectively represent the number of the olaquindox haptens coupled on one carrier protein OVA and BSA; in each olaquindox artificial antigen prepared, the value of m or n is not unique and has some changes.
The carrier protein may be Bovine Serum Albumin (BSA), Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH), or other carrier proteins.
(3) Identification of artificial antigen:
the ultraviolet scanning and SDS-PAGE identification are adopted to obtain: the coupling was successful.
Ultraviolet scanning scheme: BSA (OVA), OLA-A and OLA-A-BSA (OVA) were formulated to have a concentration of 1-5 mg/mL-1And (3) measuring the absorbance of the solution within the range of 200-400 nm, establishing an ultraviolet scanning map, and comparing the absorption curves of each solution to identify whether the synthesis is successful.
SDS-PAGE electrophoresis protocol: selecting concentrated gel with volume fraction of 5%, selecting separation gel with volume fraction of 10%, loading 10 μ L per well, concentrating gel voltage of 75V, separating gel voltage of 100V, dyeing with Coomassie brilliant blue for 1h, decolorizing for 4 times, and analyzing by gel imager.
In the ultraviolet scanning spectrum, the maximum absorption wavelength of the OLA-A-BSA (OVA) solution is changed compared with that of the BSA (OVA) solution, and SDS-PAGE shows that the electrophoretic band of the conjugate has hysteresis than that of a single protein, and the molecular weight of the conjugate is larger than that of the single protein, thereby indicating that the coupling is successful.
Comparative example 1
The olaquindox artificial antigen is synthesized by a conventional method, and the specific steps are as follows:
(1) synthesis of olaquindox hapten
Accurately adding 2.106g of olaquindox and 1.6g of succinic anhydride into a three-neck round-bottom flask, adding 80mL of pyridine, refluxing at 115 ℃ for 4h, evaporating the pyridine under reduced pressure, adding 60mL of ice distilled water and 2mol L into the rest mixture-1Adjusting the pH value to 2.0-3.0 by HCl, and standing overnight at 4 ℃. Carrying out vacuum filtration, washing with ice distilled water for 3 times, and then carrying out vacuum filtration to obtain a light yellow powdery substance, namely OLA-HS;
(2) synthesis of olaquindox artificial antigen
14.528mg OLA-HS is dissolved in 0.8mL DMF, 4.603mg NHS and 8.253mg DCC are added, and the mixture is stirred at room temperature in the dark for reaction for 10h and then 2000 r.min-1Centrifuging for 10min, and collecting supernatant as solution c.
20mg OVA (or BSA) was dissolved in 5mL of 0.01 mol.L-1In Phosphate Buffered Saline (PBS) at pH 7.4, solution b was obtained. 0.6mL of solution c was added dropwise to the slowly stirred solution b at 4 ℃ and the reaction was stirred overnight at 4 ℃. Transferring into dialysis bag at 0.01 mol/L the next day-1The precipitate was centrifuged off after dialysis for 2d in Phosphate Buffered Saline (PBS) at pH 7.4 to give a crosslinked product designated OLA-HS-OVA or OLA-HS-BSA.
(2) Identification of artificial antigen:
ultraviolet scanning and SDS-PAGE electrophoresis are adopted to identify the coupling effect.
Ultraviolet scanning scheme: BSA (OVA), OLA-HS and OLA-HS-BSA (OVA) were formulated at concentrations of1～5mg·mL-1And (3) measuring the absorbance of the solution within the range of 200-400 nm, establishing an ultraviolet scanning map, and comparing the absorption curves of each solution to identify whether the synthesis is successful.
SDS-PAGE electrophoresis protocol: selecting concentrated gel with volume fraction of 5%, selecting separation gel with volume fraction of 10%, loading 10 μ L per well, concentrating gel voltage of 75V, separating gel voltage of 100V, dyeing with Coomassie brilliant blue for 1h, decolorizing for 4 times, and analyzing by gel imager.
In the ultraviolet scanning spectrum, the maximum absorption wavelength of an OLA-HS-BSA (OVA) solution is changed compared with that of a BSA (OVA) solution, and SDS-PAGE shows that an electrophoresis strip of the conjugate has a hysteresis phenomenon compared with a single protein strip, and the molecular weight of the conjugate is larger than that of the single protein, thereby indicating that the coupling is successful.
Example 2
Determination of antiserum titers:
respectively immunizing BALB/C mice with the artificial antigens prepared in example 1 and comparative example 1, emulsifying the artificial antigen by Freund's complete adjuvant for the first immunization, emulsifying, injecting, metering to 250 μ g/mouse, boosting every 21 days, 3 times for boosting, emulsifying by incomplete adjuvant for boosting, metering to 150 μ g/mouse, cutting tail of the mouse after 14d (days) of boosting to collect blood for measuring the titer of multiple antiserum, diluting the serum by confining liquid and measuring the titer of the antiserum by ELISA method, using the OD of the mouse serum before immunization as negative control, and using the OD of the positive serum as negative control450nmValue and negative serum OD450nmThe dilution at which the value ratio was greater than 2.1 was the antiserum titer, and the results are shown in Table 1. Finally, the terminal immunization is carried out by direct intraperitoneal injection of artificial antigen, and the immunization amount is 300 mug/mouse.
The titer measurement adopts an indirect ELISA method, and the specific experimental steps are as follows:
a. coating: respectively using the artificial antigen OLA-A-OVA in example 1 or the artificial antigen OLA-HS-OVA in comparative example 1 as a coating antigen, pH 9.6, 0.05 mol.L-1CBS is coating buffer solution, and the concentration of the coating source is 10 mug.mL-1Coating amount of 100. mu.L/well, 37Coating for 2h in the middle, and washing the plate for 4 times by PBST washing liquor;
b. and (3) sealing: adding 250 mu L/hole of blocking solution, incubating for 30min at 37 ℃, and washing the plate for 4 times by PBST washing liquor;
c. adding antiserum: antiserum 10000r min obtained by collecting blood from mice-1Centrifuging for 5min, sucking 10 μ L, adding into 2mL of blocking solution (initial dilution multiple is 200 times), diluting 11 gradients and 1 negative control with blocking solution multiple ratio, repeating each gradient for 4 times at 100 μ L/well, incubating at 37 deg.C for 1h, and washing the plate with PBST washing solution for 4 times;
d. adding an enzyme-labeled secondary antibody: after the reaction is finished, washing the plate for 4 times by PBST, diluting the goat anti-mouse secondary antibody marked by HRP by 10000 times, adding the enzyme label plate, incubating for 1h at 37 ℃, and washing the plate for 4 times by PBST washing liquor;
e. adding a substrate reaction solution: adding TMB substrate buffer solution, 100 μ L/hole, and incubating at 37 deg.C for 15 min;
f. end reading: 2 mol. L is added-1Sulfuric acid 50 u L/hole, with ELISA reader read OD450nmThe value is obtained.
TABLE 1 results of antiserum titer determination in example 1 and comparative example 1
Immunity antigen Detection of antigens Antiserum potency
OLA-A-BSA example 1 OLA-A-OVA EXAMPLE 1 614400
OLA-HS-BSA comparative example 1 OLA-HS-OVA COMPARATIVE EXAMPLE 1 102400
The antiserum titer determination results in table 1 show that the antiserum titer of example 1 is higher, and the artificial antigen prepared in example 1 has a conjugation effect, so that the artificial antigen is more stable, the characteristic structure of olaquindox can be better exposed, the antigen specificity is stronger, and the preparation of the monoclonal antibody with strong specificity is facilitated.
Example 3
Preparation of olaquindox monoclonal antibody
(1) Mouse immunization:
selecting female mice with age of 6-8 weeks and weight of 18-20 g BALB/C. Pressurizing and fully mixing and emulsifying the prepared immunogen (OLA-A-BSA) and an equivalent volume Freund's complete adjuvant by a syringe, injecting the mixture at multiple points at the abdomen and the armpit, wherein the dosage is 100-450nmThe dilution at which the ratio of the value to the negative serum is greater than 2.1 is the antiserum titer. When the titer is not obviously increased any more, the cell fusion is carried out after 3d of the final immunization. In the immune process, Freund complete adjuvant is used for the first immunization, Freund incomplete adjuvant is used for boosting immunization, adjuvant is not used for the final immunization, and immunogen injection immunization is directly performed.
The titer measurement adopts an indirect ELISA method, and the specific experimental steps are as follows:
a. coating: OLA-A-OVA is used as coating antigen, pH is 9.6, 0.05 mol.L-1Carbonate Buffer (CBS) is coating buffer with the concentration of coating source being 10 mug. multidot.mL-1The coating amount is 100 mu L/hole, and after coating is carried out for 2h at 37 ℃, the PBST washing solution washes the plate for 4 times;
b. and (3) sealing: adding 250 mu L/hole of blocking solution, incubating for 30min at 37 ℃, washing the plate for 4 times by PBST, and adding 300 mu L of PBST in each hole;
c. adding antiserum: antiserum 10000r min obtained by collecting blood from mice-1Centrifuging for 5min, sucking 10 μ L, adding into 2mL of blocking solution (initial dilution multiple is 200 times), diluting 11 gradients and 1 negative control with blocking solution multiple ratio, repeating at 100 μ L/well and 4 gradients, incubating at 37 deg.C for 1h, and washing the plate with PBST washing solution for 4 times;
d. adding an enzyme-labeled secondary antibody: washing the plate for 4 times after the reaction is finished, diluting the goat anti-mouse secondary antibody marked by HRP by 10000 times, adding the enzyme label plate, incubating for 1h at 37 ℃, and washing the plate for 4 times by PBST washing liquor;
e. adding a substrate reaction solution: adding TMB substrate buffer solution, 100 μ L/hole, and incubating at 37 deg.C for 15 min;
f. end reading: 2 mol. L is added-1Sulfuric acid 50 u L/hole, with ELISA reader read OD450nmThe value is obtained.
(2) Cell fusion and culture:
after 3 days of non-immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 1500) method, and the specific steps are as follows:
a. bleeding an eyeball of a mouse after non-immunization, collecting serum, centrifuging and sucking supernatant for later use, pulling a neck to kill, putting the mouse into 70% alcohol for 3-5 min, taking a spleen of the mouse under an aseptic condition, shearing the mouse into pieces by using an aseptic operation, putting the cut pieces into an aseptic bowl mill for milling, blowing and suspending cells by using an RPMI-1640 basic culture medium, passing through a 200-mesh cell screen to obtain a splenic cell suspension, and counting the cells;
b. collecting SP2/0 cells (myeloma cells), wherein the growth state of the cells is required to be good, the cell activity is more than 90%, sucking cell supernatant, adding a new RPMI-1640 basic culture medium, blowing and suspending the cells, and then counting the cells;
c. mixing splenocytes with SP2/0 cells at a ratio of 5-10:1 according to cell count result, and heating at 1800 r.min-1Centrifuging for 5min, removing supernatant, adding 0.6mL PEG into the rest cells, stirring for 1min while adding PEG, standing for 1min, adding 45mL RPMI-1640 basic culture medium from slow to fast, and 1500 r.min-1Centrifuging for 5min, removing supernatant, adding selective HAT medium, plating in 96-well cell culture plate (250 μ L/well), standing at 37 deg.C and 5% CO by volume2Cultured in an incubator.
d. After 3-5 days of culture, the medium was changed to HAT medium 1 time, and on the 10 th day, the medium was changed to HT medium.
(3) Cell screening and cell strain establishment:
when the fused cells grow to cover 10-30% of the bottom area of the culture wells, taking the supernatant, and screening the antibody positive wells by using indirect ELISA, wherein the coating antigen is an OLA-A-OVA conjugate during screening, and OVA and BSA are used as negative controls. The screened positive reaction wells were further analyzed for antibody detection sensitivity by competitive ELISA. And (3) continuously cloning the hybridoma cells with good sensitivity for 3-4 times by using a limiting dilution method to obtain the hybridoma cell strain.
After the hybridoma cell strain is subjected to expanded culture, on one hand, the cell strain can be used for ascites preparation and monoclonal antibody purification and application; on the other hand, the established hybridoma cell strain can be transferred into a cell cryopreservation tube and placed into liquid nitrogen for long-term preservation.
(4) Preparation, purification and characterization of monoclonal antibodies
The monoclonal antibody is prepared by adopting an in-animal induction method.
Selecting 6-8 weeks old healthy BALB/C mice, injecting 0.3 mL/mouse of pristane into the abdominal cavity of the BALB/C mice, and injecting the screened hybridoma cell strain cells (0.4 mL/mouse, wherein the number of cell strains per mL is 2.5 multiplied by 106～1×107And in 5-7 days, after the abdominal cavity of the mouse is obviously expanded, carrying out aseptic operation to extract ascites, and centrifuging to remove grease precipitate to obtain the ascites of the mouse.
Purifying ascites with protein A affinity chromatographic column after octanoic acid-ammonium sulfate purification, measuring ultraviolet 260nm and 280nm optical density of purified antibody with ultraviolet spectrophotometer, and calculating protein concentration with Lowry-kalokar formula to 6.4 mg/mL-1. And storing the rest purified monoclonal antibody at-70 ℃ for later use.
Example 4
Preparation of goat anti-mouse secondary antibody
Immunizing a pathogen-free sheep by taking a sheep as an immune animal and taking a murine antibody as an immunogen, and purifying serum to obtain the vaccine; or purchasing a commercial goat anti-mouse secondary antibody product;
example 5
Preparation of fluorescent microsphere labeled anti-olaquindox monoclonal antibody
(1) And (3) activation: suspending 100 μ L microsphere suspension with fluorescent dye embedded therein and modified with carboxyl functional group on surface in 900 μ L activation buffer solution at 4 deg.C at 10000r min-1Centrifuging for 10min, removing supernatant, resuspending microspheres with 1mL of activation buffer for 2 times, adding 200 μ L of activating agent, mixing, and activating with shaking at room temperature for 15 min. The microsphere suspension is purchased from Bangs Laboratories, the microspheres are microspheres with the diameter of 100-300 nm and fluorescent substances wrapped by polystyrene, the surfaces of the microspheres are connected with-COOH groups, and the fluorescent substances are complexes of europium.
(2) Coupling: mixing the above mixed solution 10000 r.min-1Centrifuging for 10min, removing supernatant, adding 100-200 μ L olaquindox monoclonal antibody (1 mg. mL)-1) The reaction was shaken at room temperature for 2 h.
(3) And (3) sealing: after the oscillation reaction, the reaction solution is treated at the temperature of 4 ℃ for 5000r min-1After centrifugation for 15min, the supernatant was removed, resuspended in blocking buffer and then blocked with shaking at room temperature for 1 h.
(4) And (3) storage: the reaction solution after the sealing is carried out at 4 ℃ for 12000 r.min-1After centrifugation for 15min, the supernatant was removed, resuspended in storage buffer and centrifuged to discard the supernatant, after which it was again suspended in storage buffer and stored at 4 ℃ in the dark.
The pH of the activation buffer is 6.0, and 0.05 mol.L-12- (N-morphine) ethanesulfonic acid buffer (MES).
The activating agent is water-soluble carbodiimide, wherein the molar mass ratio of EDC to NHS to COOH is (2-4) to (10-20) to 1, and the activating agent is diluted to a required concentration by using an activating buffer solution before use.
The pH value of the coupling buffer solution is 7.5-8.5, and the pH value is 0.05 mol.L-1Borate buffer of (1).
The blocking buffer solution contains 0.1-0.4 mol.L-1Ethanolamine, BSA (bovine serum albumin) with the mass fraction of 2-5% and a phosphate buffer solution with the pH value of 7.4.
The storage buffer solution contains 0.01 percent of N by mass fractionaN3And 0.5% by mass of BSA, pH 7.4.
Example 6
Preparation of sample conjugate pad
(1) The sample binding pad was washed with 0.5% Bovine Serum Albumin (BSA), pH 7.4, 0.1 mol. L-1Soaking in phosphate buffer solution for 2h, and drying at 37 ℃ for 2 h;
(2) diluting the stored fluorescent microsphere labeled anti-olaquindox monoclonal antibody with a storage buffer solution, soaking the treated sample binding pad in the diluted solution for 30min, and then drying in vacuum for later use.
Example 7
Preparation of nitrocellulose membranes
With 0.05 mol.L-1The olaquindox hapten-ovalbumin conjugate (OLA-a-OVA) was diluted to 100 μ g/mL in phosphate buffer at pH 7.4-1Spraying the test solution on a detection area (N) on a nitrocellulose membrane (NC membrane) by using a membrane scratching instrument, wherein the spraying amount is 1.0-2.0 mu L-cm-1(ii) a Using 0.01 mol.L-1The goat anti-mouse secondary antibody was diluted to 60. mu.g/mL with phosphate buffer solution having pH 7.4-1Spraying the solution on a quality control area (C) on the nitrocellulose membrane by using a membrane scratching instrument, wherein the spraying amount is 1.0-2.0 mu L-cm-1. The prepared nitrocellulose membrane was dried at 37 ℃ for 2h and then kept ready for use.
Example 8
Assembly of test strips
As shown in fig. 1-2, sequentially adhering and fixing a sample combination pad 1, a nitrocellulose membrane 2 and a water absorption pad 3 on a bottom plate 6 from left to right, wherein the tail end of the sample combination pad 1 is connected with the initial end of the nitrocellulose membrane 2, the tail end of the nitrocellulose membrane 2 is connected with the initial end of the water absorption pad 3, the initial end of the sample combination pad 1 is aligned with the initial end of the bottom plate 6, the tail end of the water absorption pad 3 is aligned with the tail end of the bottom plate 6, and a detection area 4 and a quality control area 5 are fixed on the nitrocellulose membrane 2; the detection area 4 is positioned at one side close to the sample combining pad 1, and the quality control area 5 is positioned at one side far away from the sample combining pad 1; and then cutting into small strips with the width of 4mm by using a machine to form test strips, and filling the test strips into a plastic card shell to form the test card. The olaquindox fluorescent microsphere immunochromatography test paper card can be dried in the dark at 4 ℃ and has a storage validity period of up to 12 months.
Example 9
The use method of the test strip comprises the following steps:
1) pretreating the sample to obtain a sample to be detected;
2) and (3) detecting by using a test strip: sucking 100 mu L of sample solution to be detected, vertically dripping the sample solution into a sample adding hole of a test paper card, starting timing when the liquid flows, reacting for 10min, and inserting the test paper card into a fluorescence detector for detection to obtain a detection result;
3) and (4) analyzing results: after the test is finished, the instrument calculates the concentration of olaquindox in the sample according to the intensity of the fluorescence signal obtained by detection through a calculation program set by the system, and gives out positive and negative judgment according to a preset threshold value. Because each batch of test strips can carry out a large number of standard curve tests in the early stage to determine the batch of standard curves, if the batch of test strips is used for detecting actual new samples, and when a detection result is calculated, calculation can be directly carried out aiming at the previously fitted standard curves, and a calculation chip in an instrument system carries out processing to obtain the concentration of the olaquindox.
Negative (-): if the result of the fluorescence detector is negative, the olaquindox content in the sample is lower than the detection limit of the test strip;
positive (-): if the result of the fluorescence detector is positive, the olaquindox content in the sample is higher than the detection limit of the test strip;
and (4) invalidation: if the quality control area does not detect the fluorescence signal value, the detection process is incorrectly operated or the test strip is failed.
Example 10
Pretreatment method for detection sample
(1) Pretreatment of pond water sample
Filtering a pond water sample by using qualitative filter paper, accurately sucking 1mL of filtered pond water, and adding 1mL of sample diluent (the sample diluent comprises 0.01 mol. L of 5% methanol by volume)-1Phosphate buffer solution with pH 7.4), mixing uniformly, sucking 100 mu L of mixed solution, adding into a sample adding hole of a test paper card, and detecting.
(2) Feed sample pretreatment
Crushing the feed purchased in the market by a crusher, sieving the crushed feed by a 60-mesh sieve, weighing 1g of the sieved feed sample, putting the feed sample into a 5mL centrifuge tube, adding 3mL of 0.01 mol.L containing 5% methanol by volume-1Shaking pH 7.4 phosphate buffer solution on vortex oscillator for 2min, and then 5000r min-1Centrifuging for 10min, carefully sucking supernatant, transferring into 1.5mL centrifuge tube again, and centrifuging at 10000 r.min-1Centrifuging for 10min, sucking 100 μ L of supernatant, and adding into sample hole of test paper card for detection.
Example 11
Determination of test strip detection limits
Respectively adding olaquindox standard substances into a water sample, wherein the concentration of the olaquindox standard substances is 1 ng/mL-1、2ng·mL-1And 5 ng. mL-1Respectively adding olaquindox standard substances into feed samples, wherein the concentration of the olaquindox standard substances is 2 ng/mL-1、5ng·mL-1And 10 ng. mL-1The result after the detection of the fluorescence detector shows that the olaquindox concentration in the water sample is 1 ng/mL-1When the concentration of olaquindox is 2 ng/mL, the result is negative-1And 5 ng. mL-1When the test strip is used, the result is positive, which indicates that the detection limit of the test strip on a water sample is 2 ng/mL-1(ii) a In the feed sample, when the olaquindox concentration is 2 ng-mL-1When the concentration of olaquindox is 5 ng/mL, the result is negative-1And 10 ng. mL-1When the result is positive, the detection limit of the test strip on the feed sample is 5 ng/mL-1。
Example 12
Test strip false positive and false negative rate test
For water sample, respectively taking olaquindox with final concentration less than 2 ng/mL-1And greater than 2 ng/mL-1Negative and positive samples of (a); for feed samples, respectively taking olaquindox with final concentration less than 5 ng/mL-1And greater than 5 ng/mL-1Negative and positive samples of (a); 100 portions of each sample, produced in 3 different batchesThe test strips are used for respectively detecting, the positive and negative rates of the test strips are calculated, the detection result is shown in table 1, wherein the ratio of the standard deviation to the average is called a variation coefficient and is marked as CV (%), and the smaller the value of the variation coefficient is, the more concentrated and stable the data are.
TABLE 1 test results
Figure BDA0002224662780000171
As can be seen from the data in the table, when 3 batches of test strips are used for detecting positive samples, the results are all positive, and when negative samples are detected, the results are all negative, which indicates that the false positive rate and the false negative rate of the test strips are both 0; the fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues, which is established by the invention, can be used for quickly detecting water samples and feed samples, and the result accuracy is high.

Claims (7)

1. A fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues is characterized by comprising a bottom plate, a sample combination pad, a nitrocellulose membrane and a water absorption pad; the sample combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the bottom plate, the tail end of the sample combination pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample combination pad is aligned with the initial end of the bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the bottom plate; the sample combination pad is embedded with an anti-olaquindox monoclonal antibody marked by fluorescent microspheres;
a detection area and a quality control area are fixed on the nitrocellulose membrane; the detection area is positioned at one side close to the sample combination pad, the quality control area is positioned at one side far away from the sample combination pad, a goat anti-mouse secondary antibody is sprayed on the quality control area, a olaquindox hapten-carrier protein conjugate is sprayed on the detection area, the olaquindox hapten-carrier protein conjugate is an olanexin hapten-ovalbumin conjugate OLA-A-OVA,
the olaquindox hapten-ovalbumin conjugate is prepared by the following steps:
(1) synthesis of olaquindox hapten
(1.1) dissolving 1mmol of olaquindox in 2-5ml of THF, adding 1.0-1.2mmol of NaH at 0 ℃, stirring for 0.5-2 hours, adding 1mmol of 5-bromo-2, 4-diene ethyl valerate, reacting for 6-8 hours at 65 ℃, and adding 30-50mLH after the reaction is completed2O, extracting with ethyl acetate, mixing organic phases, washing with saturated saline solution, spin-drying solvent, and separating by column chromatography to obtain OLA-A1，A1is-CH-COOCH2CH3；
(1.2) adding 1mmol of OLA-A1Dissolving in 8-12mL of mixed solution of methanol and water, wherein the volumes of the methanol and the water are both 5mL), adding 1-1.5mmol of lithium hydroxide, stirring, and reacting at room temperature for 1-3 h; after the reaction is completed, 1mol/L hydrochloric acid solution is used for adjusting the pH value to be 5-6, ethyl acetate is used for extraction, an organic phase is washed by saturated saline solution and then dried by anhydrous sodium sulfate, and the solvent is removed by rotary evaporation, so that a product OLA-A is-CH-COOH; the specific synthetic route is as follows:
Figure FDA0002224662770000021
(2) synthesis of olaquindox artificial antigen
Dissolving 0.04mmol of OLA-A in 0.8-1.0mL of DMF, adding 0.04mmol of N-hydroxysuccinimide and 0.04mmol of dicyclohexylcarbodiimide, stirring at room temperature in the dark for 10-12h, centrifuging at 2000r/min for 10min, and taking the supernatant as a solution a;
weighing 20mg of OVA (or BSA) and dissolving in 5mL of 0.01mmol/L phosphate buffer solution with pH 7.4 to obtain solution b;
dropwise adding the 0.6mLa solution into the solution b at 4 ℃, and stirring and reacting at 4 ℃ overnight; transferring into dialysis bag the next day, dialyzing with 0.01mmol/L phosphate buffer solution with pH of 7.4 for 2 days, centrifuging, removing precipitate to obtain crosslinked product, and naming as OLA-A-OVA or OLA-A-BSA, -A-represents-CH-COO-; the specific synthetic route is as follows:
Figure FDA0002224662770000031
2. the fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues according to claim 1, wherein the base plate is a polyvinyl chloride base plate; the sample combining pad is glass wool; the absorbent pad is absorbent paper.
3. The fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues according to claim 1, wherein the olaquindox monoclonal antibody is obtained by coupling olaquindox hapten with carrier protein, preparing artificial antigen, immunizing mice and screening, wherein the carrier protein is ovalbumin or bovine serum albumin.
4. The fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues according to claim 1, wherein the fluorescent microsphere is a microsphere with a diameter of 100-300 nm and a fluorescent substance coated with polystyrene, the surface of the microsphere is connected with a carboxyl functional group, and the fluorescent substance is a complex of europium.
5. The fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues according to claim 1, wherein the preparation of the fluorescent microsphere labeled anti-olaquindox monoclonal antibody comprises the following steps:
1) and (3) activation: suspending 100 μ L microsphere suspension with embedded fluorescent dye and modified carboxyl functional group on surface in 900 μ L activation buffer solution at 4 deg.C for 10000r min-1Centrifuging for 10min, removing supernatant, resuspending microspheres with 1mL of activation buffer for 2 times, adding 200 μ L of activating agent, mixing, and activating with shaking at room temperature for 15 min; the microsphere is a microsphere with the diameter of 100-300 nm and a fluorescent substance wrapped by polystyrene, the surface of the microsphere is connected with-COOH groups, and the fluorescent substance is a europium complex;
2) coupling: mixing the above mixed solution 10000 r.min-1Centrifuging for 10min, removing supernatant, adding 100-200 μ L of 1 mg/mL-1Oscillating and reacting the olaquindox monoclonal antibody for 2 hours at room temperature;
3) and (3) sealing: after the oscillation reaction, theThe reaction solution is 5000 r.min at the temperature of 4 DEG C-1Centrifuging for 15min, removing supernatant, resuspending with blocking buffer solution, and sealing at room temperature for 1 hr;
4) and (3) storage: the reaction solution after the sealing is carried out at 4 ℃ for 12000 r.min-1Centrifuging for 15min, removing supernatant, resuspending with storage buffer solution, centrifuging, discarding supernatant, suspending with storage solution again, and storing at 4 deg.C in dark place;
the pH of the activation buffer is 6.0, and 0.05 mol.L-12- (N-morpholine) ethanesulfonic acid buffer (MES);
the activating agent is water-soluble carbodiimide, wherein the molar mass ratio of EDC to NHS to COOH is (2-4) to (10-20) to 1;
the pH value of the coupling buffer solution is 7.5-8.5, and the pH value is 0.05 mol.L-1The borate buffer of (1);
the blocking buffer solution contains 0.1-0.4 mol.L-1Ethanolamine, BSA (bovine serum albumin) with the mass fraction of 2-5% and a phosphate buffer solution with the pH value of 7.4;
the storage buffer solution contains 0.01 percent of N by mass fractionaN3And 0.5% by mass of BSA, pH 7.4.
6. The fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues of claim 1, wherein the preparation of the sample binding pad comprises the following steps:
1) the sample binding pad was washed with 0.5% Bovine Serum Albumin (BSA), pH 7.4, 0.1 mol. L-1Soaking in phosphate buffer solution for 2h, and drying at 37 ℃ for 2 h;
2) diluting the stored anti-olaquindox monoclonal antibody marked by the fluorescent microspheres with a storage buffer solution, soaking the sample bonding pad treated in the step 1) in the diluted solution for 30min, and then drying in vacuum for later use; the storage buffer solution contains 0.01 percent of NaN by mass fraction3And 0.5% by mass of BSA, pH 7.4.
7. An application of a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues in detection of olaquindox, which is characterized in that the test strip is the test strip of any one of claims 1 to 6, and comprises the following steps:
1) pretreating the sample to obtain a sample to be detected;
2) and (3) detecting by using a test strip: sucking 100 mu L of sample solution to be detected, vertically dripping the sample solution to be detected on a test strip sample wafer bonding pad, starting timing when the liquid flows, reacting for 10min, and inserting the test strip into a fluorescence detector for detection to obtain a detection result;
3) and (4) analyzing results: after the test is finished, the instrument calculates the concentration of the olaquindox in the sample according to the intensity of the fluorescence signal obtained by detection, and gives out positive and negative judgment according to a preset threshold value.
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