CN114195179B - Method for detecting bacillus cereus by non-diagnostic double-antibody sandwich enzyme-linked immunosorbent assay - Google Patents
Method for detecting bacillus cereus by non-diagnostic double-antibody sandwich enzyme-linked immunosorbent assay Download PDFInfo
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Abstract
The invention discloses a method for detecting bacillus cereus by double-antibody sandwich ELISA with non-diagnostic purpose, which utilizes the synthesis of Cu/CeO 2 The nanoparticle acts as a mimic enzyme, replacing HRP and secondary antibody label (conjugate), which has horseradish peroxidase (HRP) activity. Further, monoclonal antibody against B.cereus is used as capture antibody (coating antibody), polyclonal antibody against B.cereus is used as detection antibody, cu/CeO 2 The nano-particles are used as the secondary antibodies marked by the mimic enzyme, and a sandwich ELISA analysis method of B.cereus is established, and the method has the advantages of high sensitivity, good specificity, low cost and simple operation, and is used for rapid, high-throughput and low-cost detection of bacillus cereus in food.
Description
Technical Field
The invention relates to the technical field of detection of bacillus cereus, in particular to a method for detecting bacillus cereus by double-antibody sandwich ELISA with non-diagnostic purpose.
Background
Bacillus cereus (b. Cereus) is widely found in nature and is a conditional pathogen that causes food poisoning with two different symptoms, diarrhea and vomiting. Cereus is well suited for different environments such as soil, plants, insects and mammals. Because of these habits, it is often separated from the food. The pollution rate of 3 foods such as milk, cooked food and vegetables is reported to be higher than 30%. Food poisoning events caused by bacillus cereus are seriously threatening food safety and even seriously threatening human life safety.
Currently, methods for detecting B.cereus include a culture method, a PCR method and an immunoassay method. The culture method is a "gold standard" method of B.cereus identification, which comprises culture separation, selective enrichment, biochemical identification and serological identification, but the detection time generally needs 2 to 3 days. The PCR method is aimed at specific fragment DNA of the genus B.cereus, and has the characteristics of high sensitivity and short detection time. It often does not eliminate the complex procedures of enrichment, cell disruption and nucleic acid extraction. And for b.cereus population species, false positives may result due to the high degree of conservation and similarity in 16S rRNA gene sequences.
Although the detection method of food-borne pathogenic bacteria is continuously developed, the enzyme-linked immunosorbent assay (ELISA) is a common method for detecting microorganisms at present by virtue of the simplicity, specificity, sensitivity and high flux without large-scale instruments. It is noted, however, that the ability of conventional ELISA to convert target concentrations to color changes depends on the native enzyme, such as HRP or ALP. However, the high cost and stringent storage conditions prevent their use. The nano-enzyme has the potential possibility of replacing natural enzyme due to the characteristics of low cost, convenient preparation, good stability and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting bacillus cereus by double-antibody sandwich enzyme-linked immunosorbent assay without diagnosis.
A first object of the present invention is to provide a Cu/CeO 2 And (3) nanoparticles.
A second object of the present invention is to provide a Cu/CeO 2 A method for preparing nano particles.
The third object of the present invention is to provide Cu/CeO prepared by the preparation method 2 And (3) nanoparticles.
The fourth object of the present invention is the Cu/CeO 2 The application of the nano-particles in preparing an enzyme-linked immunosorbent assay kit or performing an enzyme-linked immune reaction.
The fifth object of the invention is to provide an enzyme-labeled secondary antibody.
A sixth object of the present invention is to provide any one of the Cu/CeO 2 The application of the nano-particles or the simulated enzyme-labeled secondary antibodies in the construction of an ELISA detection method and/or an ELISA kit.
The seventh object of the invention is to provide a method for detecting bacillus cereus by double-antibody sandwich ELISA without diagnosis.
The eighth object of the invention is to provide a kit for detecting bacillus cereus by double-antibody sandwich ELISA.
The invention uses bacillus cereus B.cereus (ATCC 14579, presented by the microorganism research institute of Guangdong province) to respectively immunize 8-week-old BALB/c mice and 2.5kg New Zealand white rabbits, and obtains monoclonal and polyclonal antibodies of the bacillus cereus B.cereus through 5 times of normal immunizations. Simultaneous synthesis of Cu/CeO 2 The nanoparticle acts as a mimic enzyme, replacing HRP with a secondary antibody label (conjugated), which has horseradish peroxidase (HRP) activity. Monoclonal antibody of B.cereus is used as a capture antibody, polyclonal antibody of B.cereus is used as a detection antibody, and Cu/CeO is used as a detection antibody 2 The nanoparticle is used as a mimic enzyme to mark a secondary antibody, a sandwich ELISA analysis method of B.cereus is established, and the LOD is 1.7X10 2 CFU/mL，R 2 ＝0.993。
The invention firstly claims a Cu/CeO 2 Nanoparticles of Cu/CeO 2 The nanoparticles were prepared as follows: ce (NO) 3 ) 3 ·6H 2 O、CuCl 2 ·2H 2 Adjusting the pH value of the glycol solution of O and PVP by acetic acid, and performing hydrothermal reaction to obtain a precursor; calcining the precursor to obtain the product.
Preferably, ce (NO 3 ) 3 ·6H 2 O、CuCl 2 ·2H 2 The dosage ratio of O to PVP is 300-500: 1-20: 100 to 600.
More preferably, ce (NO 3 ) 3 ·6H 2 O、CuCl 2 ·2H 2 The dosage ratio of O to PVP is 434:10:300.
preferably, the pH of the reaction system is 2.0 to 6.0.
More preferably, the pH of the reaction system is 4.0.
More preferably, the hydrothermal reaction conditions are 120-250 ℃ for 4-15 hours.
Further preferably, the hydrothermal reaction conditions are 160 ℃ for 8 hours.
More preferably, the calcination conditions are 200 to 500 ℃ for 1 to 6 hours.
Further preferably, the hydrothermal reaction conditions are 300 ℃ for 2 hours.
Further claimed is a Cu/CeO 2 Method for preparing nanoparticles, ce (NO 3 ) 3 ·6H 2 O、CuCl 2 ·2H 2 Carrying out hydrothermal reaction on the O and the ethylene glycol solution of PVP to obtain a precursor; calcining the precursor to obtain the product.
The Cu/CeO prepared by the preparation method is also claimed 2 And (3) nanoparticles.
The invention also claims the Cu/CeO 2 The application of the nano-particles in preparing an enzyme-linked immunosorbent assay kit or performing an enzyme-linked immune reaction.
The invention also claims an enzyme-labeled secondary antibody, which uses the Cu/CeO 2 The nanoparticle is coupled to a secondary antibody. The Cu/CeO 2 Nanoparticles as mimic enzymes replacing HRP and secondary antibody labels(conjugation) with horseradish peroxidase (HRP) activity
Preferably, the secondary antibody is goat anti-rabbit IgG.
Also claimed is any of the Cu/CeO' s 2 The application of the nano-particles or the simulated enzyme-labeled secondary antibodies in the construction of an ELISA detection method and/or an ELISA kit.
A method for detecting bacillus cereus by non-diagnostic double-antibody sandwich ELISA is also claimed, wherein the simulated ELISA secondary antibody is used as an ELISA secondary antibody, a monoclonal antibody for resisting bacillus cereus is used as a coating antibody, and a polyclonal antibody for resisting bacillus cereus is used as a detection antibody.
Preferably TMB and H 2 O 2 The mixed liquid of (2) is a color developing liquid.
More preferably, the method comprises the steps of:
the monoclonal antibody coats the ELISA plate;
washing the ELISA plate;
blocking redundant sites in the wells of the enzyme-labeled plate;
washing and drying the ELISA plate;
loading a sample to be tested on an ELISA plate, and incubating;
washing the ELISA plate;
the polyclonal antibody is loaded on an ELISA plate and incubated;
washing the ELISA plate;
the enzyme-labeled secondary antibody is loaded on an enzyme-labeled plate and incubated;
washing the ELISA plate;
loading the color development liquid on an ELISA plate for color development reaction;
the absorbance of the sample at 652nm was measured.
More preferably, the monoclonal antibody-coated ELISA plate is coated with 1.25-10.0. Mu.g/mL of the monoclonal antibody, and the monoclonal antibody-coated ELISA plate is fully reacted at 25-40 ℃ at 50-150. Mu.L/hole.
Further preferably, the monoclonal antibody-coated ELISA plate is coated with 2.5. Mu.g/mL of the monoclonal antibody, 100. Mu.L/well, and reacted well at 4 ℃.
More preferably, the sample to be measured is loaded in an amount of 50 to 150. Mu.L/well.
Even more preferably, the sample to be tested is loaded at 100 μl/well.
More preferably, after the sample to be detected is loaded on the ELISA plate, the incubation condition is that the incubation is carried out for 30-60 min at 25-40 ℃.
Further preferably, after the sample to be tested is loaded on the ELISA plate, the incubation condition is that the incubation is performed for 40min at 37 ℃.
More preferably, the polyclonal antibody loading is from 50 to 150. Mu.L/well at a concentration of from 0.5 to 16. Mu.g/mL.
Even more preferably, the polyclonal antibody loading of 2.0. Mu.g/mL is 100. Mu.L/well.
More preferably, after the polyclonal antibody is loaded on the ELISA plate, the incubation condition is that the polyclonal antibody is incubated at 25-40 ℃ for 30-60 min.
Further preferably, after the polyclonal antibody is loaded onto the ELISA plate, the incubation conditions are 37℃for 40min.
More preferably, the loading amount of the enzyme-labeled secondary antibody is 50-150 mu L/hole, and the loading amount is 1-10 mu g/mL.
Even more preferably, the loading of 5. Mu.g/mL of the second enzyme-labeled antibody is 100. Mu.L/well.
More preferably, after the enzyme-labeled secondary antibody is loaded on the enzyme-labeled plate, the incubation condition is that the incubation is carried out for 30-60 min at 25-40 ℃.
Further preferably, after the enzyme-labeled secondary antibody is loaded on the enzyme-labeled plate, the incubation condition is that the incubation is performed for 30min at 37 ℃.
More preferably, the loading amount of the color-developing solution is 50 to 150. Mu.L/well.
Even more preferably, the loading of the developer is 100. Mu.L/well.
More preferably, after the color development liquid is applied to the ELISA plate, the incubation condition is that the incubation is carried out for 0 to 14min at the temperature of 5 to 45 ℃.
Further preferably, after the color development liquid is applied to the ELISA plate, the incubation condition is that the incubation is performed at 35 ℃ for 8min.
More preferably, the drying ELISA plate is obtained by inverting the ELISA plate and drying at 25-40 ℃ for 0.5-2 h.
Further preferably, the dried ELISA plate is dried for 1h at 37 ℃ with the ELISA plate inverted.
More preferably, the wash plate is a PBST wash plate 1-5 times, 100-350 μl/well, and then the plate is spun or patted dry.
Further preferably, the wash microplate is a PBST wash microplate 2 times, 300. Mu.L/well, and then the microplate is spun.
More preferably, the temperature of the color reaction is 5 to 45 ℃.
Further preferably, the temperature of the color reaction is 35 ℃.
More preferably, the pH of the color-developing solution is 2 to 8.
Further preferably, the pH of the color developing solution is 4.
More preferably, the TMB concentration in the color-developing solution is 5 to 45mM.
Further preferably, the concentration of TMB in the color-developing solution is 25mM.
More preferably, H in the color-developing solution 2 O 2 The concentration of (2.5-20 mM).
Further preferably, H in the color-developing solution 2 O 2 Is 10mM.
More preferably, the solvent of the color development solution is PBS buffer.
More preferably, the reaction time for the color development is 0 to 14min.
Further preferably, the reaction time for the color development is 8min.
Preferably, the monoclonal antibody is prepared as bacillus cereus immunized mice.
Preferably, the polyclonal antibody is prepared by immunizing rabbits with Bacillus cereus.
Preferably, the coating concentration of the coating antibody is 1.25 to 10.0. Mu.g/mL, and the concentration of the detection antibody is 0.5 to 16. Mu.g/mL.
Preferably, the coating concentration of the coating antibody is 2.5. Mu.g/mL, and the concentration of the detection antibody is 2.0. Mu.g/mL.
A kit for detecting bacillus cereus by double-antibody sandwich ELISA is also claimed, which contains the simulated enzyme-labeled secondary antibody, a monoclonal antibody for resisting bacillus cereus and a polyclonal antibody for resisting bacillus cereus.
Preferably, the monoclonal antibody against bacillus cereus is immobilized on a solid support.
Preferably, the color development liquid is TMB and H 2 O 2 Is a mixed solution of (a) and (b).
More preferably, the pH of the color-developing solution is 2 to 8.
Further preferably, the pH of the color developing solution is 4.
More preferably, the TMB concentration in the color-developing solution is 5 to 40mM.
Further preferably, the concentration of TMB in the color-developing solution is 25mM.
More preferably, H in the color-developing solution 2 O 2 The concentration of (2.5-20 mM).
Further preferably, H in the color-developing solution 2 O 2 Is 10mM.
More preferably, the solvent of the color development solution is PBS buffer.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes the synthesis of Cu/CeO 2 The nanoparticle acts as a mimic enzyme, replacing HRP and secondary antibody label (conjugate), which has horseradish peroxidase (HRP) activity. Further, monoclonal antibody against B.cereus is used as capture antibody (coating antibody), polyclonal antibody against B.cereus is used as detection antibody, cu/CeO 2 The nano-particles are used as the secondary antibodies marked by the mimic enzyme, and a sandwich ELISA analysis method of B.cereus is established, and the method has the advantages of high sensitivity, good specificity, low cost and simple operation, and is used for rapid, high-throughput and low-cost detection of bacillus cereus in food.
Drawings
FIG. 1 is an antibody specificity.
FIG. 2 is a Cu/CeO diagram 2 The results were characterized.
FIG. 3 is a Cu/CeO diagram 2 The synthesis conditions are Cu/CeO 2 Influence of nanoparticle preparation.
FIG. 4 is a diagram of Cu/CeO 2 Steady state kinetic analysis of catalytic TMB.
FIG. 5 is a graph of external conditions versus Cu/CeO 2 Influence of catalytic properties.
FIG. 6 is a standard curve of Bacillus cereus based on the simulated enzyme double antibody sandwich method.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 preparation of Bacillus cereus B.cereus antibodies
1. Experimental method
1. Preparation of immunogens
Bacillus cereus (ATCC 14579, available from the Ministry of microorganisms, guangdong) was washed with a culture solution from Centrohol, inactivated with 4% (m/m) paraformaldehyde for 1 hour, washed with sterile saline, resuspended in sterile saline, and placed at-80℃until use.
2. Animal immunization protocol
(1) Experimental animals: 4 BALB/c mice of 6-8 weeks old and 1 New Zealand white rabbit of 2.5-3.0 kg were selected for immunization.
(2) Emulsification: emulsifying the same volume of immunogen and adjuvant, wherein the immunogens of immunized mice and rabbits respectively comprise 10 8 And-10 9 Individual cells, primary immunization was emulsified with complete adjuvant and booster immunization was emulsified with incomplete adjuvant. After the emulsification is completed, abdominal cavity and subcutaneous multipoint injection are adopted.
(3) The immunization method comprises the following steps: mice and rabbits were immunized every two weeks according to a specific immunization protocol.
(4) Serum titer detection: on day 7 after the end of the third immunization, the titers of the antisera were determined by indirect ELISA. Wherein, the mice take blood through tail breaking and the rabbits take blood through the veins at the ear.
The specific steps of serum titer detection are as follows:
1) Inactivated Bacillus cereus B.cereus at 10 with coating solution 7 CFU/mL cells were diluted and coated on 96-well ELISA plates, 100. Mu.L/well, and overnight at 37 ℃. The ELISA plate was taken out the next day, washed twice with PBST, and dried on absorbent paper.
2) After sufficient washing, the redundant sites are blocked by blocking liquid, 200 mu L/hole is placed in a 37 ℃ incubator for incubation for 2 hours, and then taken out for drying for standby.
3) The mouse antiserum or rabbit antiserum was added to the ELISA plate by gradient dilution, and negative control, 100. Mu.L/well, incubated at 37℃for 40min, and washed 5 times for drying.
4) HRP goat anti-mouse or goat anti-rabbit IgG at 1:5000 was added to the ELISA plate at 100. Mu.L/well, incubated at 37℃for 30min and washed 5 beats.
5) The color development liquid (TMB: h 2 O 2 =1:1, v/v) was added to the elisa plate at 100 μl/well and incubated for 10min at 37 ℃. 50. Mu.L of 10% (v/v) H was added 2 SO 4 The reaction was terminated and absorbance at 450nm was measured with a microplate reader. Wherein the color developing solutions TMB and H 2 O 2 The concentration of (2) is 2.5mM, and both are prepared by sodium citrate buffer with pH of 3.5 and 0.01M, and are respectively stored at 4 ℃ before use, and are uniformly mixed in a volume of 1:1 when in use.
(5) Antibody preparation:
1) Polyclonal antibodies: on day 7 after the end of the last booster immunization, the rabbits were anesthetized by intraperitoneal injection of 10% chloral hydrate, and rabbit antisera were prepared from whole blood of the rabbits.
2) Monoclonal antibodies: day 7 after the end of the last booster immunization, about 10 was injected into the abdominal cavity of the mice with the optimal titers 8 Individual cells were subjected to impact immunization without any adjuvant. Spleen cells were selected for fusion with murine myeloma cells. The hybridoma cells with good specificity and high titer are screened (including positive and negative screening) by an indirect ELISA method, and the screened cells are subjected to 5 rounds of limiting dilution to obtain the single cell strain with the best antibody production. The obtained single cell clone was amplified and injected into the abdominal cavity of a 10-12 week old BALB/c mouse to produce ascites.
(6) Purification and preservation of antibodies: purifying the obtained rabbit antiserum and ascites by octanoic acid-saturated ammonium sulfate method, dialyzing to obtain polyclonal antibody (16 mg/mL) and monoclonal antibody (10 mg/mL) of Bacillus cereus B.cereus, measuring concentration, and packaging at-20deg.C.
2. Experimental results
The results of the titer tests performed on the mouse antisera and the rabbit antisera taken from the seventh day after the third booster immunization are shown in the attached table 1, and the results show that the titers increase with the increase of the number of immunization. The monoclonal antibody is prepared by utilizing rabbit antiserum to prepare a polyclonal antibody, and generating a hybridoma secreting the monoclonal antibody after the spleen cells of the mice 2 are fused with the mouse myeloma cells.
TABLE 1 results of immunotitres
EXAMPLE 2 Bacillus cereus B.cereus antibody specificity
1. Experimental method
Dilution of inactivated Bacillus cereus B.cereus with coating solution (10) 7 CFU/mL) and strains of other species (10) 8 CFU/mL) (B.thuringiensis.metacinn-active S.aureus, E.coli O157: H7, L.monocytogenes, V.paralytic, B.thuringiensis, B.lichenifermis, B.megaterium, B.mycolide, metacinn-resisitive S.aureus, S.enteritidis, S.tyrphinium and S.pullulium) was then coated on 96-well ELISA plates at 100. Mu.L/well overnight at 37 ℃. The ELISA plate was taken out the next day, washed twice with PBST, and dried on absorbent paper.
The remaining steps are identical to the antibody titer determination step.
2. Experimental results
The antibody dilution with absorbance of about 1.0-1.5 was selected, and the specificity of the monoclonal antibody was observed, and the results showed that only b.cereus was reacted when the monoclonal antibody was diluted 32-fold, and the absorbance decreased with increasing dilution, indicating that the monoclonal antibody prepared had high specificity, as shown in fig. 1 in detail.
EXAMPLE 3 monoclonal and polyclonal antibody concentration
1. Experimental method
The concentration of the coated antibody (monoclonal antibody prepared in example 1) and the concentration of the detection antibody (polyclonal antibody prepared in example 1) were optimized by a checkerboard assay, and the specific experimental procedure was as follows:
1) The coated antibodies (monoclonal antibodies prepared in example 1) were diluted with coating solution to concentrations of 10, 5, 2.5, 1.25. Mu.g/mL, respectively, and coated on 96-well ELISA plates at 100. Mu.L/well overnight at 4 ℃. The ELISA plate was taken out the next day, washed twice with PBST, and dried on absorbent paper.
2) After sufficient washing, the redundant sites are blocked by blocking liquid, 200 mu L/hole is placed at 37 ℃ for incubation for 2 hours, and then taken out for drying for standby.
3) The sample was diluted to a concentration of 10 with phosphate buffer solution having a pH of 7.4.0.01M 7 CFU/mL was added to the ELISA plate and negative control was performed, 100. Mu.L/well, incubated at 37℃for 40min, and washed 5 times for drying.
4) The detection antibody (polyclonal antibody prepared in example 1) was diluted to a concentration of 16, 8, 4, 2,1, 0.5, 0. Mu.g/mL, 100. Mu.L/well with phosphate buffer at pH 7.4.0.01M, incubated at 37℃for 40min and washed 5 beats.
5) Sheep anti-mouse or sheep anti-rabbit IgG-HRP was diluted 1:5000 with phosphate-Tween 20 buffer pH 7.4.01M, 100. Mu.L/well was added to the ELISA plate, incubated at 37℃for 30min and washed 5 beats.
6) The color development liquid (TMB: h 2 O 2 =1:1, v/v) was added to the elisa plate at 100 μl/well and incubated for 10min at 37 ℃. 50. Mu.L of 10% (v/v) H was added 2 SO 4 The reaction was terminated and absorbance at 450nm was measured with a microplate reader. Wherein the color developing solutions TMB and H 2 O 2 The concentration of (2) is 2.5mM, and both are prepared by sodium citrate buffer with pH of 3.5 and 0.01M, and are respectively stored at 4 ℃ before use, and are uniformly mixed in a volume of 1:1 when in use.
Since the natural enzyme (goat anti-mouse or goat anti-rabbit IgG) requires strong acid to terminate the reaction, TMB+ is changed into TMB 2+ The color changes from blue to yellowThe maximum absorbance was 450nm.
2. Experimental results
As shown in Table 2, the results of the chessboard measurement were shown to give 4 pairs of P/N > 8, wherein the concentration of the coating antibody (monoclonal antibody prepared in example 1) was 10, 5, 2.5 and 1.25. Mu.g/mL, and the concentration of the corresponding detection antibody (polyclonal antibody prepared in example 1) was 2.0. Mu.g/mL. Wherein the concentration of the coating antibody (monoclonal antibody prepared in example 1) and the concentration of the detection antibody (polyclonal antibody prepared in example 1) were 2.5. Mu.g/mL and 2.0. Mu.g/mL, respectively, the corresponding P/N values were the largest.
Table 2 chessboard assay results:
EXAMPLE 4Cu/CeO 2 Synthesis and characterization of nanoparticles
1. Experimental method
434mg Ce (NO) 3 ) 3 ·6H 2 O，10mg CuCl 2 ·2H 2 Adding 20mL of ethylene glycol (PVP is 100-200 mg, 300mg and 400-600 mg) into 300mg of PVP, adjusting the pH value of a reaction system to 4.0 by acetic acid, synthesizing a precursor through hydrothermal reaction (160 ℃ C., 8 h), and calcining at 300 ℃ C. For 2h to obtain a finished product Cu/CeO 2 Nano particles.
Cu/CeO 2 The surface functional groups are characterized by Scanning Electron Microscopy (SEM), transmission Electron Microscopy (TEM), X-ray energy spectrum analysis (EDS), X-ray diffraction (XRD), fourier transform infrared absorption spectrometer (FTIR), ultraviolet-visible spectrophotometry (UV-Vis) and other instruments.
2. Experimental results
Cu/CeO 2 The characterization result of the nano-particle finished product is shown in figure 3, and figure 2A shows Cu/CeO 2 Is a nano particle with 200nm diameter and regular and uniform shape, and has rough surface. TEM (FIG. 2B) shows Cu/CeO 2 Is a hollow structure.
The characteristic peak of Cu in EDS spectrum is evident (FIG. 2C), indicating Cu elementThe element is successfully doped in CeO 2 Is a kind of medium.
EDS elemental profiles (fig. 2D through 2G) show a uniform distribution of elements including Cu, ce, and O.
Cu/CeO 2 XRD (fig. 2H) of the nanoparticles clearly showed bragg reflection angles at 28.5, 33.1, 47.5, 56.3 °, matching the (111), (200), (220), (311) crystal planes, which is comparable to CeO 2 Is consistent in the body-centered cubic structure (JCPDS No. 34-0394). In addition, diffraction peaks of the (222), (400) and (331) crystal planes are also apparent.
In the FTIR spectrum (FIG. 2I), at 3446cm -1 And 1061cm -1 The absorption peak at the location was attributed to-OH stretching and bending vibrations, respectively, at 1618cm -1 And 1382cm -1 The absorption peaks at c=o and Ce-O-Ce stretching vibrations, respectively. Thus, the FTIR spectrum results indicate that the composition is in Cu/CeO 2 The surface of the nanoparticle has the presence of-COOH.
EXAMPLE 5 dosage of PVP and pH of reaction System vs. Cu/CeO 2 Influence of nanoparticles
1. pH value of reaction system is to Cu/CeO 2 Influence of nanoparticles
1. Experimental method
434mg Ce (NO) 3 ) 3 ·6H 2 O，10mg CuCl 2 ·2H 2 Adding 20mL of ethylene glycol into 300mg of PVP, adjusting the pH value of a reaction system to 2.0, 4.0 and 6.0 by acetic acid, synthesizing a precursor by hydrothermal reaction (160 ℃ for 8 hours), and calcining at 300 ℃ for 2 hours to obtain a finished product Cu/CeO 2 And (3) nanoparticles.
2. Experimental method
Cu/CeO 2 The result of the synthesized product of the nanoparticles is shown in FIG. 3A, and the synthesized Cu/CeO is obtained when the pH of the system is 4 2 The nanoparticles were uniform in size and small in diameter (200 nm).
2. Dosage of PVP to Cu/CeO 2 Influence of nanoparticles
1. Experimental method
434mg Ce (NO) 3 ) 3 ·6H 2 O，10mg CuCl 2 ·2H 2 O, an amount of PVP was added to 20mL of ethylene glycol (PVPThe dosage is 100-200 mg, 300mg and 400-600 mg), the pH value of the acetic acid regulating reaction system is 4.0, the precursor is synthesized through hydrothermal reaction (160 ℃ for 8 h), and the final product Cu/CeO is obtained after further calcination at 300 ℃ for 2h 2 And (3) nanoparticles.
2. Experimental method
Cu/CeO 2 The result of the synthesized product of the nano-particles is shown in FIG. 3B, and when PVP is added in an amount of 300mg, the synthesized Cu/CeO 2 The nano particles are complete and uniform.
EXAMPLE 6Cu/CeO 2 Determination of peroxidase Activity of nanoparticles
1. Experimental method
200. Mu.L of the Cu/CeO prepared in example 4 at 1mg/mL was monitored in a 0.01M Tris-HCl buffer system at pH 4.0 for 10min 2 The nanoparticles catalyzed different concentrations of 500. Mu.L TMB (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 and 0.45 mM) and 500. Mu.L 0.1mM H 2 O 2 The change in absorbance at 652nm was calculated by Michaelis-Menten equation max Obtaining the concentration of the substrate TMB and the maximum reaction speed V max And the relation and the double derivative equation of (2) to finally obtain the Michaelis constant K m 。Cu/CeO 2 Peroxidase activity of nanoparticles by Cu/CeO 2 Mie constant K of catalysis of substrate TMB by nanoparticles m To evaluate, where K m Calculated by the following Michaelis-Menten equation.
Wherein: k (K) m The value is Mi's constant, V max Is the reaction rate of the enzyme when saturated with substrate, [ S ]]Is the substrate concentration.
2. Experimental results
The results are shown in FIG. 4, and the steady-state dynamics experiment shows that Cu/CeO 2 The peroxidase of the nanoparticle catalyzes the process. The substrate concentration and initial rate in the curve (FIGS. 4A and 4B) show a typical Michaelis-Menten reaction. Cu/CeO based on Lineweaver-Burk plot and with TMB as substrate 2 Mie constant K of nanoparticles m Is 0.33 mM, lower than the literature (Zhu, h., quan, z., hou, h., cai, y., liu, w.,&enzyme constant (K) of horseradish peroxidase in Liu, Y (2020) Acolorimetric immunoassay based on cobalt hydroxide nanocages as oxidase mimics for detection of ochratoxin A. Analytical Chimica Acta,1132,101-109.10.1016/j. Aca.2020.07.068.) m =0.43 mM), indicating Cu/CeO 2 The nanoparticles have higher affinity for the substrate of TMB.
EXAMPLE 7 Secondary antibody and Cu/CeO 2 Coupling of nanoparticles
1. Experimental method
10mg/mL of Cu/CeO prepared in example 4 2 The nanoparticles were sonicated in 1mL Tris-HCl (0.01 mol/L, pH 7.4) buffer, then Cu/CeO was activated with 0.4mol/L EDC and 0.1mol/L NHS 2 The carboxyl groups on the nanoparticles were conjugated with commercial 1mg/mL goat anti-mouse IgG and finally the excess sites were blocked with 50. Mu.L of 10% (m/m) BSA. After blocking was complete, excess unbound antibody and BSA was removed by centrifugation (6000 rpm,10 min) and resuspended in 1mL Tris-HCl buffer for use.
2. Experimental results
The product was measured by UV-Vis and the results are shown in FIG. 2J. Cu/CeO 2 The carboxyl on the surface of the nanoparticle can be directly used for loading Ab 2 (commercial goat anti-mouse IgG) without modification. In FIG. 2J, ab 2 The absorption peak of (C) is 280nm. Cu/CeO 2 The nanoparticle appears to peak at about 370 nm. Ab (Ab) 2 (commercial goat anti-mouse IgG) with Cu/CeO 2 After nanoparticle coupling, cu/CeO was detected 2 Absorption peaks of nanoparticles and proteins, demonstrating Ab 2 Successful modification on Cu/CeO 2 On the nano particles, cu/CeO is prepared 2 -Ab 2 。
EXAMPLE 8Cu/CeO 2 Optimization of nanoparticle catalysis conditions
In the following experiment, the buffer system was 0.01mol/L PBS, cu/CeO 2 The concentration was 5mM, the reaction system was 200. Mu.L, 20. Mu.L of Cu/CeO 2 ，50μL H 2 O 2 And 50. Mu.L TMB, 80. Mu.L PBS buffer, whereinH 2 O 2 And TMB were both formulated with PBS buffer.
1. Reaction temperature vs. Cu/CeO 2 Influence of catalytic Activity
1. Experimental method
20μL Cu/CeO 2 80. Mu.L of PBS buffer at pH 6.0, 50. Mu.L of 10mM H 2 O 2 And 50. Mu.L of 10mM TMB, respectively, were reacted at 5, 10, 15, 20, 25, 30, 35, 40℃for 10 minutes, 3 in each group in parallel, and the absorbance at 652nm was measured at the end of the reaction.
2. Experimental results
As shown in FIG. 5A, the experimental results show that Cu/CeO increases with increasing temperature 2 The light absorption intensity of the color generated by catalyzing TMB at 652nm is gradually enhanced, reaches the highest value after 35 ℃, and does not rise any more when the temperature is continuously increased, thus Cu/CeO 2 The temperature of the catalytic TMB is preferably 35 ℃.
2. pH of reaction system is relative to Cu/CeO 2 Influence of catalytic Activity
1. Experimental method
20μL Cu/CeO 2 mu.L of PBS buffer of different pH (2, 3, 4, 5, 6, 7, 8), 50. Mu.L of 10mM H 2 O 2 And 50. Mu.L of 10mM TMB, were reacted at 35℃for 10 minutes, 3 in each group in parallel, and the absorbance at 652nm was measured at the end of the reaction.
2. Experimental results
As shown in FIG. 5B, the pH of the reaction system is 2-4, cu/CeO 2 The absorption intensity of the color generated by catalyzing TMB at 652nm is gradually increased, reaches the highest value after the pH value is 4.0, and starts to suddenly decrease when the pH value is continuously increased, thus Cu/CeO 2 The preferred reaction system for catalyzing TMB has a pH of 4.
3. TMB concentration vs Cu/CeO 2 Influence of catalytic Activity
1. Experimental method
20μL Cu/CeO 2 80. Mu.L of PBS buffer at pH 4, 50. Mu.L of 10mM H 2 O 2 And 50. Mu.L of TMB at different concentrations (5, 10, 15, 20, 25, 30, 35, 40 mM) were reacted at 35℃for 10min, 3 replicates per group, and the end of reaction was assayed for 652nm absorbanceLight intensity.
2. Experimental results
As shown in FIG. 5C, the Cu/CeO concentration increased with TMB concentration 2 The absorption intensity of the color produced by catalyzing TMB at 652nm is gradually increased, reaches the highest value at 25mM, and does not rise any more when the concentration is continuously increased, thus Cu/CeO 2 The preferred reaction system for catalyzing TMB has a TMB concentration of 25mM.
4. Reaction time vs. Cu/CeO 2 Influence of catalytic Activity
1. Experimental method
20μL Cu/CeO 2 80. Mu.L of PBS buffer at pH 4, 50. Mu.L of 10mM H 2 O 2 And 50. Mu.L of 25mM TMB were reacted at 35℃for different times (0, 2, 4, 6, 8, 10, 12, 14 min), 3 in parallel each, and the absorbance at 652nm was measured at the end of the reaction.
2. Experimental results
As shown in FIG. 5D, the experimental results show that Cu/CeO increased with the increase of the reaction time 2 The light absorption intensity of the color generated by catalyzing TMB at 652nm is gradually enhanced, reaches the highest value at 8min, and does not rise any more when the reaction time is continued, thus Cu/CeO 2 The preferred reaction time for catalyzing TMB is 8min.
To sum up, cu/CeO 2 The reaction of catalyzing TMB is greatly influenced by external conditions, the optimal reaction temperature is 35 ℃, the pH is 4, the TMB concentration is 25mM, and the reaction time is 8min.
Example 9 double antibody Sandwich assay for determining Bacillus cereus B.cereus Standard Curve based on mimic enzyme
1. Experimental method
1. Coating: the ELISA plate was coated with 2.5. Mu.g/mL of the monoclonal antibody prepared in example 1, 100. Mu.L/well, and overnight at 4 ℃.
2. Washing: the ELISA plate was washed 2 times with PBST, 300. Mu.L/well, and then dried.
3. Closing: the excess sites were blocked by incubation with 3% (mass fraction) skimmed milk powder at 200 μl/well for 2h at 37 ℃.
4. And (3) drying: the liquid in the ELISA plate is thrown off forcefully, and is beaten to be dry on the absorbent paper, and the ELISA plate is inverted and dried for 1h at 37 ℃.
5. Sample: mu.L of sample (from 10 to B.cereus with PBS) was added to each well 8 CFU/mL was diluted 3-fold, 13 gradients total, as detection samples, and a PBS blank was set up) and incubated at 37 ℃ for 40min.
6. Washing: the elisa plate was washed 5 times with PBST, 300 μl/well, and blotted dry on absorbent paper.
7. Detection of antibodies: 2.0. Mu.g/mL of the polyclonal antibody prepared in example 1 was added to the microplate at 100. Mu.L/well and incubated at 37℃for 40min.
8. Washing: and the same as in step 6.
9. Simulating enzyme-labeled secondary antibodies: cu/CeO obtained in example 7 2 - Ab 2 The ELISA plate was added at 5. Mu.g/mL and 100. Mu.L/well and incubated at 37℃for 30min.
10. Washing: and the same as in step 6.
11. Color development: cu/CeO obtained based on example 8 2 The optimal conditions for catalyzing TMB reaction are used for preparing the required color development liquid. The color development liquid comprises two parts: TMB and H respectively 2 O 2 Solutions, both of which were prepared with phosphate buffer at pH 4.0.01M, were prepared at final concentrations of 25mM and 10mM, respectively. Before use, the mixture is preserved at 4 ℃, when in use, the mixture is mixed uniformly in a volume of 1:1, 100 mu L/hole is added into an ELISA plate, incubation is carried out for 8min at 35 ℃, and the light absorption intensity of 652nm is measured after the reaction is finished.
12. And (3) measuring: the absorbance of the sample at 652nm was measured.
Since the mimic enzyme catalyzes TMB and H as a catalyst 2 O 2 Termination with strong acid results in complete cleavage of the mimic enzyme and the production of a black material that affects the assay, and is generally not performed when catalyzed by the mimic enzyme. The absorbance at 652nm was thus determined.
2. Experimental results
Bacillus cereus B.cereus was assayed using a double antibody sandwich method based on a mimic enzyme, resulting in a linear range of 3.2X10 2 Up to 1X 10 5 CFU/mL，R 2 ＝0.993See fig. 6 for details.
Bacillus cereus b.cereus was based on the mimic enzyme double antibody sandwich method lod=1.7x10 2 CFU/mL (LOD is the average absorbance in space plus 3 times the standard deviation of the corresponding antibody concentration).
Example 10 method for determining Bacillus cereus B.cereus by double antibody sandwich method based on mimic enzyme
1. Detection method
1. Coating: the ELISA plate was coated with 2.5. Mu.g/mL of the monoclonal antibody prepared in example 1, 100. Mu.L/well, and overnight at 4 ℃.
2. Washing: the ELISA plate was washed 2 times with PBST, 300. Mu.L/well, and then dried.
3. Closing: the excess sites were blocked by incubation with 3% (mass fraction) skimmed milk powder at 200 μl/well for 2h at 37 ℃.
4. And (3) drying: the liquid in the ELISA plate is thrown off forcefully, and is beaten to be dry on the absorbent paper, and the ELISA plate is inverted and dried for 1h at 37 ℃.
5. Sample: 100. Mu.L of the sample to be tested was added to each well and incubated at 37℃for 40min.
6. Washing: the elisa plate was washed 5 times with PBST, 300 μl/well, and blotted dry on absorbent paper.
7. Detection of antibodies: 2.0. Mu.g/mL of the polyclonal antibody prepared in example 1 was added to the microplate at 100. Mu.L/well and incubated at 37℃for 40min.
8. Washing: and the same as in step 6.
9. Simulating enzyme-labeled secondary antibodies: cu/CeO obtained in example 4 2 - Ab 2 The ELISA plate was added at 5. Mu.g/mL and 100. Mu.L/well and incubated at 37℃for 30min.
10. Washing: and the same as in step 6.
11. Color development: the color development liquid comprises two parts: TMB and H respectively 2 O 2 The solutions were each prepared with phosphate buffer at pH 4.0.01M and the final concentrations were 25mM and 10mM, respectively. Before use, the mixture is preserved at 4 ℃, when in use, the mixture is mixed uniformly in a volume of 1:1, 100 mu L/hole is added into an ELISA plate, and the mixture is incubated at 35 ℃ for 8min.
12. And (3) measuring: the absorbance of the sample at 652nm was measured.
2. Result determination
The absorbance at 652nm was positive. If the concentration of Bacillus cereus B.cereus is less than 1.7X10 2 CFU/mL, since the captured sample is too small to cause a sufficient signal change, was judged negative.
Example 11 double antibody Sandwich method based on mimic enzyme kit for determination of Bacillus cereus B.cereus
1. Composition of the composition
Monoclonal antibody prepared in example 1, ELISA plate, washing solution (PBST), 3% skimmed milk powder, polyclonal antibody prepared in example 1, cu/CeO prepared in example 7 2 -Ab 2 Color development solutions (TMB and H) 2 O 2 10mM PBS at a solution pH of 4.0 at a concentration of 25mM and 10mM, respectively).
2. Application method
As in example 10.
Comparative example method for determining bacillus cereus B.cereus by double antibody sandwich method based on mimic enzyme
1. Detection method
The same as in example 10, but the enzyme label plate is coated by using the polyclonal antibody as a coating antibody, and the monoclonal antibody as a detection antibody is added into the enzyme label plate for incubation.
2. Result determination
No absorbance at 652nm was found, which was not applicable to B.cereus detection.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of protection of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (3)
1. The method comprises the following steps ofThe method for detecting bacillus cereus by using non-diagnostic double-antibody sandwich ELISA is characterized in that a simulated enzyme-labeled secondary antibody is used as the enzyme-labeled secondary antibody, the secondary antibody is goat anti-rabbit IgG, a monoclonal antibody for resisting bacillus cereus is used as a coating antibody, a polyclonal antibody for resisting bacillus cereus is used as a detection antibody, and the simulated enzyme-labeled secondary antibody is Cu/CeO 2 The nano particles are coupled with secondary antibody, and the Cu/CeO is prepared by 2 The nanoparticles were prepared as follows: ce (NO) 3 ) 3 •6H 2 O、CuCl 2 •2H 2 Carrying out hydrothermal reaction on an ethylene glycol solution of O and PVP to obtain a precursor, wherein the hydrothermal reaction condition is 160 ℃ for 8 hours; calcining the precursor to obtain the catalyst, wherein the condition of the calcining reaction is 300 ℃ for 2 hours; calculated by mass, ce (NO 3 ) 3 •6H 2 O、CuCl 2 •2H 2 The dosage ratio of O to PVP is 434:10:300, the pH of the reaction system is 4.0; TMB and H in a volume ratio of 1:1 2 O 2 The mixed solution of (2) is a chromogenic solution, the chromogenic reaction temperature is 35 ℃, the pH is 4, the TMB concentration is 25mM, and H 2 O 2 The concentration was 10mM and the reaction time was 8min.
2. The method of claim 1, wherein the coating concentration of the coating antibody is 1.25 to 10.0 μg/mL and the concentration of the detection antibody is 0.5 to 16 μg/mL.
3. A kit for detecting bacillus cereus by double-antibody sandwich ELISA is characterized by comprising the simulated enzyme-labeled secondary antibody, the monoclonal antibody for resisting bacillus cereus and the polyclonal antibody for resisting bacillus cereus as set forth in claim 1, and further comprising a chromogenic solution, wherein the chromogenic solution comprises TMB and H in a volume ratio of 1:1 2 O 2 The mixed solution of (2) is a color developing solution, the pH is 4, the TMB concentration is 25mM, and H 2 O 2 The concentration was 10mM.
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