CN107942053B

CN107942053B - Spore @ Zr4+Preparation of functional microsphere and application of functional microsphere in immunoassay

Info

Publication number: CN107942053B
Application number: CN201610497713.2A
Authority: CN
Inventors: 胡涌刚; 夏苗苗; 李政
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2016-06-30
Filing date: 2016-06-30
Publication date: 2020-06-09
Anticipated expiration: 2036-06-30
Also published as: CN107942053A

Abstract

The invention belongs to the field of biological material preparation, and particularly relates to preparation of spore @ Zr4+ as a functional microsphere and application of the spore @ Zr4+ in immunoassay. Processing spores by a biochemical method to prepare suspension microspheres with high stability and good dispersibility, and then carrying out Zr⁴⁺Ions are adsorbed on the surface of the spores and are applied to the field of immunodetection. The invention is characterized in that bacterial spores cultured for 7 days are collected, centrifuged, washed and treated by ultrasonic waves, and then treated by trypsin liquid and spore coat removing treatment liquid to obtain bacterial spore microspheres with smooth surfaces and outer walls and spore coats of the bacterial spores removed, and the microspheres are used for adsorbing Zr⁴⁺Spore @ Zr prepared by ion⁴⁺Functional microspheres. The functional microsphere has the advantages of good mechanical strength, uniform particle size, proper specific gravity, simple preparation process, low cost, suitability for high-throughput detection and environmental friendliness. The invention also discloses spore @ Zr⁴⁺The application of the microsphere in the immune detection of the infection of the mycobacterium bovis.

Description

Spore @ Zr4+Preparation of functional microsphere and application of functional microsphere in immunoassay

Technical Field

The invention belongs to the field of preparation of immunoassay materials, and particularly relates to spore @ Zr⁴⁺A preparation method of functional microspheres and application thereof in immunoassay.

Background

The immunoassay technology is an analysis method based on antigen-antibody specific reaction. Due to the advantages of high specificity, high sensitivity, low cost and the like, the technology is an important technology for analyzing specific proteins or other substances. However, conventional immunoassays still have certain drawbacks.

Taking enzyme-linked immunosorbent assay (ELISA) as an example, the method has high cost and high labor intensity, and is obviously limited in detection dynamic range and detection sensitivity. As a new immunoassay method, the immunoassay microsphere detection technology breaks through the inherent limitations of the traditional method and develops rapidly^1-8. Compared with the traditional immunoassay technology, the immunoassay method based on the microspheres has the advantages that: (1) the specific surface area is large. Compared with a two-dimensional planar structure, the specific surface area of the microsphere is greatly increased, and the combination of a receptor and the surface of the microsphere is facilitated; (2) the binding rate is moreAnd (4) the method is quick. The immunoassay method based on the microspheres is carried out in a suspended liquid phase, so that the influence of surface tension, space effect and the like on reaction kinetics during macromolecular detection of a solid phase carrier is overcome, and the reaction is accelerated; (3) the operation is simple. The separation of the immunoassay based on the microspheres can be directly carried out by centrifugation or magnetic separation, and the separation and washing are more convenient and faster. (4) Low cost and less consumption of immunodetection samples. The microspheres are easier to produce in a large scale and at low cost, and only a trace sample volume is needed when the microspheres are used for immunoassay; (5) high throughput detection. The immune microspheres can be used for simultaneously carrying out qualitative and quantitative analysis on a plurality of different target molecules in one sample. (6) There is greater flexibility in sample analysis and data processing. According to the related research, the ideal microspheres for immunoassay have the characteristics of uniform particle size, density slightly larger than water, quick reaction, easy separation, easy washing and the like⁹. However, many microspheres reported previously for immunoassay have the disadvantage of too large particle size or too large density, and are difficult to be used in immunoassay. For example, the microspheres currently commercialized are mostly polymer-synthesized, and the microspheres tend to aggregate during the reaction with the sample rather than being suspended in the supernatant, thereby reducing the diffusion efficiency of the microparticles and further affecting the detection efficiency^9,10. In recent years, the research team of the applicant develops a novel biological method for preparing highly monodisperse microspheres with various functional groups (amino, carboxyl and hydroxyl) on the surfaces by using spores of bacillus and proves that the microspheres have remarkable application potential in specific fields^11-13. The spore-based microspheres have good physicochemical properties such as good mechanical strength, uniform particle size, proper specific gravity and the like, which just meet the ideal conditions of the microspheres for immunoassay⁹. Meanwhile, compared with the traditional microspheres, the biological microspheres also have the advantages of simple preparation, low cost, suitability for high-throughput detection, environmental friendliness and the like. The biggest advantage is that the preparation method can be used for mass production by industrialized bacterial fermentation, and the preparation method is low in cost and environment-friendly. The spore-based microspheres have the advantages that the spore-based microspheres are applied to other wider fieldsIt is possible.

For immunoassays, the current methods for preparing immunomicrospheres generally involve coupling an antigen or antibody to a functionalized microsphere using conventional coupling reagents (N-hydroxysuccinimide ester, water-soluble carbodiimide)¹⁴. However, this coupling procedure is complicated, costly, time consuming, unsuitable for trace amounts of protein and requires specialized technical training. Therefore, it is urgently needed to find a method for assembling capture protein, which is simple to operate, high in performance and low in cost. Over the past decades, metal ion functionalized microspheres have been enriched for biomolecules (polypeptides or proteins)¹⁵Enzyme immobilization¹⁶And environmental pollution abatement¹⁷The application reported in the aspects of the technology is gradually attracting attention. Generally, commercial microspheres are pre-modified with functional groups such as amino, carboxyl and phosphate groups on their surface as chelating agents for fixing metal ions during synthesis^18-20(ii) a However, chemical modification processes are often complex, time consuming and environmentally hazardous.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel method for simply and rapidly preparing highly monodisperse functional microspheres.

The applicant for the first time uses Zr⁴⁺As a model to construct metal ion functionalized materials for immunoassay. The invention provides an innovative method, namely, the highly monodisperse biological functional microspheres are prepared by using spores of bacillus and are applied to diagnosis of bovine tuberculosis. In the strategy proposed by the present invention, metal ions (e.g. Zr)⁴⁺) Can be directly combined on the surface of the spore without additional modification process. This is because some inherent functional groups (such as carboxyl and amino) exist on the surface of spore biological microsphere prepared by the invention. Obviously, metal ions are fixed on the spore-based functional microspheres to form the spores @ Zr⁴⁺Compared with the traditional method for preparing the microspheres for immunoassay, the method is simpler, more convenient, quicker and safer. Accordingly, the metal ion is used as a binding medium for immobilizing capture protein, which has advantages of easy operation, environmental friendliness, and low cost. Spore @ Zr prepared⁴⁺The microspheres adopt two immunoassay strategies to detect the mycobacterium bovis infection: (1) capture of antigen protein MPB83 through Zr⁴⁺Immobilization of spore @ Zr⁴⁺On microspheres, followed by the addition of M.bovis-infected serum to react with immune microspheres, DyLight^TM649-goat anti-bovine IgG was subsequently added to participate in the secondary immune response, and finally the amount of antibody in M.bovis-infected serum was analyzed by flow cytometry. (2) In a second strategy, the Applicant demonstrated that antibodies could also be immobilized on spores @ Zr⁴⁺The surface of the microspheres was used to detect bovine IFN-. gamma.released from peripheral blood mononuclear cells caused by infection with M.bovis. After double-antibody sandwich immunoreaction, horseradish peroxidase (HRP) -H is adopted₂O₂-Amplex Red fluorescent reaction System^21,22Bovine IFN-gamma was subjected to immunoassay. Finally these assays were used for clinical diagnosis of M.bovis infection. The invention overcomes the defects of complex operation, high energy consumption, unfriendly environment, uneven density and particle size and the like of the traditional preparation of functional microspheres. The invention fixes metal zirconium ions on the surface of the treated spores to form the spores @ Zr⁴⁺The microspheres are applied to immunoassay.

In order to achieve the purpose, the invention adopts the following technical scheme:

collecting bacterial spores cultured for 7 days, centrifuging, washing, performing ultrasonic treatment, treating with trypsin solution and de-spore coat buffer solution (degassing buffer), and adsorbing zirconium ions to obtain spore microspheres with smooth surface and high dispersibility and hydrophobicity⁴⁺The microspheres are used for detecting antibodies in bovine mycobacterium infection serum and bovine IFN-gamma fluorescence immunoassay.

Specifically, the technical scheme of the invention is as follows:

spore @ Zr prepared by using bacterial spores⁴⁺A method of functionalizing a microsphere comprising the steps of:

(1) culturing bacillus and collecting spores: respectively storing 200 μ L of Bacillus amyloliquefaciens or Bacillus megaterium strain in 50% glycerol at-20 deg.CAdding the mixture into sterilized 5mL LB liquid culture medium, and placing the mixture in a 37 ℃ temperature-controlled shaking table for culturing overnight at 180rpm to activate the strains; when OD is reached₆₀₀When the concentration is 0.6, sucking 200 mu L of the spore, adding the liquid into a cooled LB solid culture medium plate, uniformly coating the liquid by using a glass rod, collecting the spores cultured for 7 days, washing the spores by using deionized water, centrifuging the spores and repeating the steps for 4 to 5 times;

(2) spore treatment: after the collected spores of bacillus amyloliquefaciens and spores of bacillus megatherium are re-suspended by double distilled water, carrying out ultrasonic treatment for 5min each time by an ultrasonic crusher under the ice bath condition, cooling the spore liquid at an ultrasonic interval of 5min, and carrying out centrifugal washing for 3-4 times after the ultrasonic output intensity is 7-8, the frequency is 20% -30% and the ultrasonic time is 20 min; collecting the precipitated spores, resuspending with 15-20 mL of 1% trypsin solution, and shaking at 80rpm in a shaker at 37 deg.C overnight; centrifuging to remove trypsin solution, adding 15-20 mL of coating removing agent (coating Buffer), and reacting for 2h under magnetic stirring at 70 ℃; centrifuging the treated spores to remove supernatant, cleaning the spores for 5 times by using double-distilled water, re-suspending the spores, taking a small amount of the spores for counting, inactivating the rest at 121 ℃ for 30min under high pressure to inactivate the spores, namely spore stock solution, and storing the spore stock solution at 4 ℃ for later use;

(3) the spores adsorb zirconium ions: 1mL of the treated spore stock solution was placed in an EP tube, centrifuged at 12000rpm for 2 minutes, the supernatant was discarded, and 1mL of 0.5mol/L Zr was added⁴⁺The spores are resuspended by the ionic solution, blown evenly to disperse the spores fully, the EP tube is put into a shaking table to oscillate at 100rpm for 1h, the supernatant is removed by centrifugation, the solution is washed three times by double distilled water and stored in a refrigerator at 4 ℃ for standby;

wherein:

the Bacillus is a strain which is publicly issued by China center for type culture collection and has a collection number of CCTCC AB 92052, and the Bacillus amyloliquefaciens is a strain which is publicly issued by China center for type culture collection and has a collection number of CCTCC AB 2013062.

The applicant provides a method for the application of functional microspheres prepared by using bacterial spores, wherein the application comprises the following two directions: :

1. spore @ Zr prepared by the invention⁴⁺The application of the microspheres in detecting the antibody of the mycobacterium bovis infected serum comprises the following steps: the method comprises the following specific steps:

(1) 100 μ L of 1.2X 10 was taken¹⁰particles mL^–1Spore @ Zr⁴⁺Microspheres and 10. mu.g mL^-1The MPB83 antigen protein reacts for 20min at 37 ℃ to couple the MPB83 antigen protein to the spore @ Zr⁴⁺On the surface of the microspheres;

(2) taking out the reaction centrifuge tube, adding 500 μ L PBST into each tube after centrifugation to elute the immune microspheres, mixing uniformly, standing for 3min, centrifuging to remove the eluent, and repeatedly washing for 3 times;

(3) uses 15% sheep serum solution as a sealant to treat spores coupled with MPB83 antigen protein @ Zr⁴⁺Sealing the microspheres;

(4) taking out the reaction centrifuge tube, and repeating the step (2);

(5) adding 200uL of serum to be detected to ensure that the antibody in the serum and the antigen protein generate specific immunoreaction to form spore @ Zr⁴⁺A microsphere-antigen-antibody complex;

(6) taking out the reaction centrifuge tube, and repeating the step (2);

(7) 200. mu.L of 0.25. mu.g mL of the solution was added^-1Dylight^TM649 labeled goat anti-bovine IgG was subjected to a second immunoreaction, which was followed by sufficient washing to allow Dylight not bound to the microsphere surface^TM649-IgG was eluted well;

(8) after completion of the elution, the microspheres after completion of the immunoreaction were resuspended in PBS buffer, and Dylight labeled on the surfaces of the microspheres was detected by flow cytometry^TM649 fluorescence signal intensity.

2. Spore @ Zr prepared by the invention⁴⁺The application of the microsphere in bovine IFN-gamma fluorescence immunoassay comprises the following specific steps:

(1) 1mL of the solution was taken at a concentration of 3.30X 10⁹spores/mL @ Zr⁴⁺Centrifuging the microspheres to remove supernatant, adding 5ug/mL anti-bovine IFN-gamma monoclonal antibody, mixing, and shaking at 37 deg.C and 160r/min in a table type constant temperature shaking tableIncubating for 1 h;

(2) taking out the reaction tube, centrifuging to remove the supernatant, washing with 1mL of PBST for 3 times, and carrying out blocking reaction for 2h with 1mL of mixed blocking agent containing 5% Bovine Serum Albumin (BSA) and 5% skim milk (skim milk);

(3) after blocking, the microspheres were washed 3 times with 1mL of PBST and resuspended in 1mL of PBS buffer containing 0.1% BSA;

(4) take 6.60X 10⁷Adding the immune spore microspheres into a 2mL centrifuge tube, washing for 3 times by using 500 mu L PBST, and centrifuging to remove the supernatant; adding 100 μ L of standard substance with different concentration gradients or plasma sample to be detected, oscillating, and reacting in a table type constant temperature shaking table at 37 deg.C and 180r/min for 30 min;

(5) taking out the reaction centrifuge tube, adding 500 μ L PBST into each tube after centrifugation to elute the immune microspheres, mixing uniformly, standing for 3min, centrifuging to remove the eluent, and repeatedly washing for 3 times;

(6) 100 μ L of 0.50 μ g mL was added to each tube^-1The anti-bovine IFN-gamma polyclonal antibody is uniformly oscillated and reacts for 30min in a table type constant temperature shaking table at 37 ℃ under the condition of 180 r/min;

(7) taking out the reaction centrifuge tube, and repeating the step (2);

(8) adding 100 μ L of 2.50 μ g/mL horseradish peroxidase-labeled goat anti-rabbit IgG into each tube, shaking, mixing, and reacting at 37 deg.C for 30min at 180r/min in a table-type constant temperature shaking table;

(9) taking out the reaction centrifugal tube, and repeating the step (2) to obtain immune spore microspheres;

(10) 50 μ L of 0.20mmol/L H₂O₂And 0.10mmol/L Amplex Red and the immune spore microsphere obtained in the step (9) are mixed evenly, and 50 mu L of 0.20mmol/LH is added into the centrifuge tube directly₂O₂And 0.10mmol/LAmplex Red as a blank. Placing the centrifuge tube in a table type constant temperature shaking table in a dark place, reacting for 20min at 37 ℃ and 180r/min, centrifuging, absorbing 50 mu L of supernatant, and detecting the fluorescence signal of each sample by using a fluorescence and chemiluminescence detector;

(11) drawing a standard curve: and (3) taking the concentration of the standard substance as an abscissa, detecting the fluorescence intensity value as an ordinate, and manually drawing or drawing a standard curve by using software. The concentration of the sample to be detected can be checked on the standard curve according to the fluorescence intensity value of the sample to be detected;

(12) and (5) judging the result of the bovine tuberculosis diagnosis. Calculating the average fluorescence signal intensity read by detecting the PPD of the poultry and PPD of the cattle by using the method to stimulate plasma of each animal;

the calculation formula is as follows:

the positive PPD-stimulated plasma of cattle/PPD-stimulated plasma of fowl detects fluorescence signal intensity more than or equal to 1.71;

(ii) the fluorescence signal intensity of bovine purified tuberculin/avian purified bindin is < 1.71; wherein:

the Bacillus is a strain Bacillus megaterium (Bacillus megaterium) which is publicly issued by China center for type culture collection and has a collection number of CCTCC AB 92052, and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a strain which is publicly issued by China center for type culture collection and has a collection number of CCTCC AB 2013062.

Drawings

FIG. 1: treated spores and spores @ Zr⁴⁺And (5) microsphere characterization. FIG. 1A and FIG. 1B are transmission electron micrographs of treated Bacillus amyloliquefaciens and Bacillus megaterium, respectively; FIG. 1C shows Zr adsorption by spores⁴⁺FTIR spectra before and after the ion. Description of reference numerals: black line represents spore adsorption of Zr⁴⁺FTIR spectrum before ion, red line shows spore adsorption Zr⁴⁺FTIR spectrum after; panel D in fig. 1 is XPS energy spectrum of treated spores; e picture is spore @ Zr⁴⁺XPS energy spectrum of (a); f is Zr in E⁴⁺XPS peak profiles of elements.

FIG. 2: spore @ Zr⁴⁺The functional microsphere is used as a suspension carrier in antibody immunoassay. FIG. 2 is a graph A showing the intensity of detection signals of negative sera; the B graph in FIG. 2 is the detection signal intensity of the positive serum; the C plot in FIG. 2 is a linear plot of the detection of standard positive sera over the 1: 1000-and 20000-fold dilution range; FIG. 2 is a graph D showing bovine tuberculosis positive serum and bovine infectious nasal airThe test result chart of the control of the positive serum of angiitis, the positive serum of babesiosis bovis, the positive serum of foot-and-mouth disease, the positive serum of Brucella, the positive serum of Eperythrozoon bovis and the negative serum of tuberculosis of cattle.

FIG. 3: spore @ Zr⁴⁺The functional microsphere is used as a suspension carrier in antigen immunoassay. FIG. 3 is a graph A showing the spectra of the positive and negative samples, and the inset shows the colorimetric results of the positive and negative samples; FIG. 3B shows that the amount of IFN-. gamma.in the bovine IFN-. gamma.standard is 0.1ng ml^-1To 20ng mL^-1A linear plot of detection over a range of concentrations; in FIG. 3, the C diagram is based on spore @ Zr⁴⁺The specificity research of the bovine IFN-gamma immunoassay method. Illustration of the C-plot label in fig. 3: the abscissa represents 3.0ng mL in order from left to right^-1Bovine IFN-. gamma.3.0 ng mL^-1Bovine IFN-. gamma. +1.0ng mL^-1Bovine IFN- α,3.0ng mL^-1Bovine IFN-. gamma. +5.0ng mL^-1Bovine IFN- α,3.00ng mL^-1Bovine IFN-. gamma. +1.0ng mL^-1Bovine IFN- β,3.0ng mL^-1Bovine IFN-. gamma. +5.0ngmL^-1Bovine IFN- β, Control (-), 1.0ng mL^-1Bovine IFN- α,5.0ng mL^-1Bovine IFN- α,1.0ng mL^-1Bovine IFN- β,5.0ng mL^-1Bovine IFN- β.

Detailed Description

Example 1 (preparation example)

In the embodiment, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a strain which is publicly issued by the China center for type culture collection and has a collection number of CCTCC AB2013062) and Bacillus megaterium (Bacillus megaterium) is a strain which is publicly issued by the China center for type culture collection and has a collection number of CCTCC AB 92052) are taken as starting materials for preparing the functional microspheres.

The method comprises the following specific steps:

(1) culturing bacillus and collecting spores: respectively taking 200 mu L of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or Bacillus megaterium (Bacillus megaterium) strain preserved in 50 percent of glycerol at the temperature of-20 ℃, adding the strain into sterilized 5mL of LB liquid culture medium, and putting the strain into a temperature-controlled shaking table at the temperature of 37 ℃ for culturing at 180rpm overnight to activate the strain; when OD is reached₆₀₀When the concentration is 0.6, sucking 200 mu L of the spore, adding the liquid into a cooled LB solid culture medium plate, uniformly coating the liquid by using a glass rod, collecting the spores cultured for 7 days, washing the spores by using deionized water, centrifuging the spores and repeating the steps for 4 to 5 times;

(2) spore treatment: and (2) after the collected spores of bacillus amyloliquefaciens and spores of bacillus megatherium are re-suspended by double distilled water, carrying out ultrasonic treatment for 5min each time by using an ultrasonic crusher under the ice bath condition, cooling the cell sap at an ultrasonic interval of 5min, carrying out ultrasonic treatment on the cell sap for 3-4 times after carrying out ultrasonic treatment for 20min, wherein the ultrasonic output intensity is 7-8 and the frequency is 20% -30%. Collecting the precipitated spores, resuspending with 15-20 mL of 1% trypsin solution, and shaking at 80rpm in a shaker at 37 deg.C overnight; centrifuging to remove trypsin solution, adding 15-20 mL of coating removing agent (coating Buffer), and reacting for 2h under magnetic stirring at 70 ℃; centrifuging the treated spores to remove supernatant, cleaning the spores for 5 times by using double-distilled water, re-suspending the spores, taking a small amount of the spores for counting, inactivating the rest at 121 ℃ for 30min under high pressure to inactivate the spores, namely spore stock solution, and storing the spore stock solution at 4 ℃ for later use;

(4) for treated spores and spores @ Zr⁴⁺And (3) carrying out characterization on the microspheres: the treated spores described in example 1 were characterized by Transmission Electron Microscopy (TEM) and treated spores and spores @ Zr⁴⁺The microspheres were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray electron spectroscopy (XPS).

The results of this example are shown in fig. 1, and transmission electron micrographs (see fig. 1, panel a, and fig. 1, panel B) of bacillus megaterium (b.amyloliquefaciens) and bacillus amyloliquefaciens (b.amyloliquefaciens) spores reveal that the treated spore surface is very smooth and oval. Bacillus amyloliquefaciens (B. amyloliquefaciens) spores and methods based on amyloliquefaciensSpore @ Zr of bacillus spore⁴⁺The functional microspheres were further analyzed by FTIR and XPS, and it was found from FTIR analysis that the surface of monodisperse spore microspheres (see FIG. 1, indicated by black lines in C diagram) carry carboxyl functional groups (RCOOH; 1654,1541,1241and 1067 cm)^-1) Peptide bond (CO-NH-; 1654, and 1541cm^-1) And amino groups (-NH)₂,and–NH； 3282,1557,669cm^-1) (ii) a Spore @ Zr⁴⁺FTIR characterization of the microspheres revealed surface locations of 3282 and 1654cm^-1Absorption peak at the site of Zr bonding⁴⁺Red-shifting to 3288 and 1652cm after ion^-1To (3). Treated monodisperse spore microspheres (edited according to the new figure number of the attached drawing, see figure 1, panel D) and spore @ Zr⁴⁺The functional microspheres (panels E and F in FIG. 1) were analyzed by XPS, respectively. From plot E in FIG. 1, plot F in FIG. 1 shows the characteristic peak for Zr3d at 182.22eV, which is greater than that for ZrOCl at 184.71eV₂.8H₂The O binding energy is slightly lower. Meanwhile, the combination of Ols and Nls can adsorb Zr on spores⁴⁺The latter increases by about 0.53eV and 1.25eV, respectively, due to Ols and the loss of Nls electron density. The characterization results of FTIR and XPS prove that the treated spore microsphere passes through amino, carboxyl and Zr on the surface of the spore microsphere⁴⁺The ions are combined by chemical adsorption or coordination.

Example 2 (application example 1)

Spore @ Zr prepared by the invention⁴⁺Application of microspheres in detection of mycobacterium bovis infected serum antibody

The method comprises the following specific steps:

(3) uses 15% sheep serum solution as blocking agent to treat spore coupled with MPB83 antigen protein@Zr⁴⁺Sealing the microspheres;

(4) and (4) taking out the reaction centrifuge tube, and repeating the step (2).

(6) taking out the reaction centrifuge tube, and repeating the step (2);

The immune microspheres prepared by the invention are used for respectively detecting the standard negative serum and the standard positive serum of the bovine tuberculosis, and the linear range of the standard positive serum with different dilution times and the cross infection among other pathogenic factors are tested, and the result is shown in figure 2. The detection signal intensity of the negative serum and the detection signal intensity of the positive serum are respectively shown in the graph A in the graph 2 and the graph B in the graph 2, and the detection signal intensity of the positive serum is obviously higher than that of the negative serum, so that the differentiation is good. After optimization of relevant conditions, the spore @ Zr prepared by the method⁴⁺The microspheres have good linearity, repeatability and specificity in detecting bovine tuberculosis. R in standard positive serum diluted 500-20000 times²The relative standard deviation of 11 replicates was 2.33% at 0.997 (results are shown in panel C of fig. 2), with a detection limit of 32000-fold dilution of standard positive serum, which is much lower than that of the conventional ELISA method (S/N3). Meanwhile, in the present example, the detection analysis was performed on bovine infectious rhinotracheitis positive serum, bovine babesiosis positive serum, foot-and-mouth disease positive serum, brucella positive serum, bovine eperythrozoon positive serum, and five positive sera (the results are shown in fig. 2D). The fluorescence signal intensity of the five infected serums and the fluorescence of bovine tuberculosis negative control serum can be known from the figureThe light value is similar and far lower than the positive control serum signal value of bovine tuberculosis, which indicates that the spores adsorb Zr⁴⁺The ions do not have cross reaction with other pathogenic bacteria when detecting bovine tuberculosis infection serum, which shows that the specificity of the method is good. The detection result of 69 clinical bovine serums is shown in figure 1 by using the kit and the commercial bovine tuberculosis antibody ELISA detection kit, and the result of figure 1 shows that the sensitivity of the kit is 71.43%, the coincidence rate is 86.95% and the specificity is 97.50%.

TABLE 1 spores @ Zr⁴⁺Comparison of results of composite microspheres and ApxIVA-ELISA for detecting clinical serum of pigs

Thus, the spore @ Zr constructed by the invention⁴⁺The microsphere has great potential and application prospect in the aspect of using the suspension chip for detecting the antibody in serum.

Example 3 (application example 2)

Spore @ Zr⁴⁺Application of microspheres in bovine IFN-gamma fluorescence immunoassay

1. Culture of Whole blood

54 parts of cow blood samples are collected from cow farms in Huanggang city, Hubei province, and heparin is added for anticoagulation. Transporting to a laboratory at room temperature and culturing within 30h after blood collection. Adding anticoagulated blood into 24-well tissue culture plate, adding 1.5mL of anticoagulated blood into 2 tubes of each cow, adding 100 μ L of avian PPD and bovine PPD into the corresponding wells, and incubating in 37 deg.C incubator for 16-24 h. Approximately 400. mu.L of the supernatant plasma was carefully pipetted for subsequent testing.

2. Bovine IFN-gamma fluoroimmunoassay procedure

(1) 1mL of the solution was taken at a concentration of 3.30X 10⁹spores/mL @ Zr⁴⁺Centrifuging the microspheres to remove supernatant, adding 5ug/mL anti-bovine IFN-gamma monoclonal antibody, mixing, and incubating in a table type constant temperature shaking table at 37 deg.C and 160r/min for 1 h;

(4) take 6.60X 10⁷The individual immune spore microspheres were added to a 2mL centrifuge tube, washed 3 times with 500. mu.L of PBST, and centrifuged to remove the supernatant. Adding 100 μ L of standard substance with different concentrations or plasma sample to be detected, shaking, and reacting in a table type constant temperature shaking table at 37 deg.C and 180r/min for 30 min;

(7) taking out the reaction centrifuge tube, and repeating the step (2);

(9) taking out the reaction centrifuge tube, and repeating the step (2);

(10) 50 μ L of 0.20mmol/L H₂O₂Mixing with 0.10mmol/L Amplex Red and the immune spore microsphere obtained in the previous step, and adding 50 μ L0.20 mmol/LH directly into the centrifuge tube₂O₂And 0.10mmol/L AmplexRed as a blank. Placing the centrifuge tube in a table type constant temperature shaking table in a dark place, reacting for 20min at 37 ℃ and 180r/min, centrifuging, absorbing 50 mu L of supernatant, and detecting the fluorescence signal of each sample by using a fluorescence and chemiluminescence detector.

(11) And (5) drawing a standard curve. And (3) taking the concentration of the standard substance as an abscissa, detecting the fluorescence intensity value as an ordinate, and manually drawing or drawing a standard curve by using software. The concentration of the sample to be detected can be checked on the standard curve through the fluorescence intensity value of the sample to be detected.

(12) And (5) judging the result of the bovine tuberculosis diagnosis. The mean fluorescence signal intensity read from PPD-stimulated plasma from birds and bovine animals tested by this method was calculated for each animal. Calculating the formula:

fluorescence signal intensity detected by positive-cattle PPD-stimulated plasma/fluorescence signal intensity detected by poultry PPD-stimulated plasma is not less than 1.71

Fluorescent signal intensity of bovine purified tuberculin/avian purified bindin <1.71

HRP-based catalyst for H₂O₂Oxidizing Amplex Red to generate resorufin with strong fluorescence property, and establishing a new material based on spore @ Zr⁴⁺And the new fluorescence immunosensing technology is successfully used for immunoassay of bovine IFN-gamma and early diagnosis of bovine tuberculosis. The graph A in FIG. 3 is a fluorescence spectrum of the positive sample and the negative sample, and it can be seen from this graph that the fluorescence intensity of the positive sample is much higher than that of the negative sample. The B plot results in fig. 3 show that: at 0.1ng mL^-1-20.0ng mL^-1The concentration of bovine IFN-gamma in the range has a good linear relation (R) with the fluorescence signal intensity value²0.998); the detection limit is 40pg mL^--1(S/N-3) (FIG. 3, panel B). The concentration of the nano-particles is 1.5ng mL^–1Bovine IFN- α and IFN- β were used to evaluate the diagnostic specificity, the results are shown in graph C in FIG. 3, the fluorescence signal value of detected bovine IFN- α and bovine IFN- β was close to that of the negative control, while the fluorescence signal value of detected bovine IFN- α and bovine IFN- γ after mixing and the fluorescence signal value of detected bovine IFN- β and bovine IFN- γ after mixing were close to the fluorescence signal intensity generated by separately detecting bovine IFN- γ of the same concentration, indicating that the method has specificity for detection of bovine IFN- γ, in order to investigate the application prospect of the present invention for diagnosis of bovine tuberculosis, 54 plasma samples stimulated with bovine PPD and PPD separately were detected and compared with a commercial Boviga mTM kit, and the results are shown in Table 2, with a coincidence rate of 87.0%, a sensitivity of 82.6%, and a specificity of 90.3%.

TABLE 2 Bovigam^TMKit and methodComparison of results of diagnosing bovine tuberculosis

The above results show that the spore @ Zr constructed by the invention⁴⁺The microsphere can successfully carry out fluorescence immunoassay on bovine IFN-gamma so as to realize early diagnosis of bovine tuberculosis, and spores @ Zr⁴⁺The microspheres have good commercial application prospects in double-antibody sandwich immunoassay.

Remarking: the information on the biomaterials, reagents and instruments used in the examples is as follows:

bacterial spores: bacillus from 7 days of culture comprising strains taken from: the Bacillus megaterium CCTCC AB 92052 and the Bacillus amyloliquefaciens CCTCC AB2013062 are both preserved in China Center for Type Culture Collection (CCTCC), belong to publicly issued biological materials, but the implementation of the invention is not limited to the biological materials.

And (3) related reagents: prokaryotic recombinant expression of bovine IFN-gamma, murine anti-bovine IFN-gamma monoclonal antibody, rabbit anti-bovine IFN-gamma polyclonal antibody and plasma samples are provided by the subject group of professor Guo Aizhen in the national emphasis laboratory of university of agriculture in Huazhong (the preparation method is shown in the subject group to issue papers Li C, Tan YD, Hu QY, Chen YY, Ma Y, Zhang GR, et al.Chin JBiomed Eng 2007; 23: 40-45), and prokaryotic recombinant expression of Mycobacterium bovis specific antigen protein MPB83 is purchased from ABConal biotechnology Limited; the test water is ultrapure water; tryptone and yeast extract were purchased from Oxoid, UK; trypsin, Amplex Red was purchased from Sigma-Aldrich, USA; sodium dodecyl sulfate, zirconium oxychloride octahydrate, 30% H₂O₂The solution and the glycerol are purchased from national pharmaceutical chemical reagent limited; 1, 4-dithiothreitol is purchased from Heifengbo Biotech, Inc.; HRP-labeled goat anti-rabbit IgG was purchased from Abbkine, usa; coomassie Brilliant blue G250 was purchased from Tianjin Wei chemical engineering, Inc.; skim milk was purchased from bidi medical devices ltd (shanghai); bovine Serum Albumin (BSA), Tween-20 from Roche, Germany; bovigam^TMA mycobacterium bovis gamma-interferon detection kit was purchased from prionieseg; bovine PPD and avian PPD were purchased from Chinese veterinary medicine institute.

Preparation of main solution:

(1) 1% trypsin solution: 1.00g of trypsin was weighed, dissolved in 0.01mol/L phosphate buffer solution (PBS solution) (usual buffer solution) having pH7.4, and then diluted to 100mL with ultrapure water.

(2) Coating removing agent (coating buffer) weighing 0.40g sodium hydroxide and 5.84 × 10^-1Sodium chloride (g), sodium dodecyl sulfate (1.00 g), dithiothreitol (1.54 g), and ultrapure water (ultrapure water) to a constant volume of 100 mL.

0.50mol/L Zr⁴⁺Solution: 16.10g of zirconium oxychloride octahydrate are weighed out and dissolved in 100mL of water.

(3) The PBS preparation method comprises the following steps: 8.00g of sodium chloride, 0.20g of potassium chloride, 3.63g of disodium hydrogen phosphate dodecahydrate and 0.24g of potassium dihydrogen phosphate, and adding ultrapure water to a constant volume of 1L of ultrapure water.

(4) The preparation method of the PBST eluent comprises the following steps: 1L of PBS solution was added with 500. mu.L of Tween-20.

(5) The preparation method of the Bradford working solution comprises the following steps: 100mg of Coomassie brilliant blue G250 was weighed out and dissolved in 50mL of 95% ethanol, 100mL of 85% phosphoric acid was added thereto, diluted with ultrapure water to a volume of 1L, and stored in a brown bottle and filtered through a filter paper before use.

Relevant instrument manufacturers and models (operating according to the instructions provided by manufacturers during actual use):

a pH meter: PB-10, Allilon, USA;

a water purifier: CASCADA IX MK2, Poll, USA;

transmission electron microscope: JEM-100CX II, Japan Electron Ltd;

fourier transform infrared spectrometer: 330FTIR, Saimer Feishol technologies, USA;

a high-speed refrigerated centrifuge: sigma 3K15, Sigma, germany;

particle size and potential analyzer: NanoZS90, malvern, uk;

plasma spectrometer: IRIS II, thermoelectric element corporation, usa;

x-ray photoelectron spectroscopy: VG M ultilab 2000, Sammer Feishell science, USA;

an ultrasonic crusher: 450D, Branson corporation, USA;

fluorescence and chemiluminescence detector: fluoroskan Ascent FL, Sammer Feishel technologies, USA;

flow cytometry: FACSCalibur, BD corporation, usa.

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Claims

1. Spore @ Zr⁴⁺The application of the functional microspheres in the preparation of immunodiagnostic reagents is characterized in that the application method comprises the following steps:

(1) culturing bacillus and collecting spores: respectively taking 200 mu L of bacillus amyloliquefaciens or bacillus megaterium strain preserved in 50% of glycerol at the temperature of-20 ℃, adding the strain into sterilized 5mL of LB liquid culture medium, and placing the strain in a 37 ℃ temperature-controlled shaking table for overnight culture at 180rpm to activate the strain; when OD is reached₆₀₀When the volume is 0.6, 200. mu.L of the solution is pipetted and added to a cooled LB solid medium plate, and the solution is spread uniformly on a glass rodCollecting spores cultured for 7 days, washing with deionized water and centrifuging, and repeating for 4-5 times;

(2) spore treatment: after the collected spores of bacillus amyloliquefaciens and spores of bacillus megaterium are re-suspended by double distilled water, carrying out ultrasonic treatment for 5min each time by an ultrasonic crusher under the ice bath condition, cooling the spore liquid at the ultrasonic interval of 5min, carrying out ultrasonic treatment on the spore liquid with the ultrasonic output intensity of 7-8 and the frequency of 20-30%, and carrying out centrifugal washing for 3-4 times after carrying out ultrasonic treatment for 20 min; collecting the precipitated spores, resuspending with 15-20 mL of 1% trypsin solution, and shaking overnight at 80rpm in a shaker at 37 ℃; centrifuging to remove trypsin solution, adding 15-20 mL of coating removing agent (coating Buffer), and reacting for 2h under magnetic stirring at 70 ℃; centrifuging the treated spores to remove supernatant, washing the spores for 5 times by using double distilled water, re-suspending the spores, taking a small amount of the spores for counting, inactivating the rest of the spores at 121 ℃ for 30min under high pressure to inactivate the spores, namely spore stock solution, and storing the spore stock solution at 4 ℃ for later use;

(3) spore adsorption of zirconium ions: taking 1mL of the treated spore stock solution, placing the treated spore stock solution in an EP tube, centrifuging at 12000rpm for 2min, discarding the supernatant, and adding 1mL of 0.5mol/L Zr⁴⁺Resuspending spores by using an ionic solution, blowing uniformly to fully disperse the spores, putting an EP tube into a shaking table, oscillating at 100rpm for 1h, centrifuging to remove supernatant, washing with double distilled water for three times, and storing in a refrigerator at 4 ℃ for later use;

wherein:

the formula of the coat removing agent (coating Buffer) in the step (2) is as follows: 0.40g of sodium hydroxide, 5.84X 10 are weighed out separately^-1Adding ultrapure water into sodium chloride (g), sodium dodecyl sulfate (1.00 g) and dithiothreitol (1.54 g) to reach the constant volume of 100 mL;

the formulation of the 1% trypsin solution was as follows: weighing 1g of trypsin, and dissolving in 100ml of PBS buffer solution with pH7.4;

zr of 0.5mol/L described in step (3)⁴⁺The solution preparation method comprises the following steps: weighing 8.056g of zirconium oxychloride octahydrate and dissolving in 50mL of deionized water;

the bacillus in the step (2) is bacillus megaterium (Bacillus megaterium) with the preservation number of CCTCC AB 92052 and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) with the preservation number of CCTCC AB 2013062;

said Zr⁴⁺The ion is derived from zirconium oxychloride octahydrate (ZrOCl)₂·8H₂O), molecular weight 322.25.