CN114045266A

CN114045266A - Anti-mellea acid monoclonal antibody and application thereof

Info

Publication number: CN114045266A
Application number: CN202111456116.2A
Authority: CN
Inventors: 徐振林; 曹雪铭; 王宇; 雷红涛; 孙远明; 罗林; 王弘; 沈玉栋
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-02-15

Abstract

The invention discloses a monoclonal antibody of anti-zymotic acid and application thereof, and an immunoassay method established based on the monoclonal antibody has high sensitivity and can supplement the requirement of low sensitivity at present. Wherein the linear detection range of the enzyme-linked immunosorbent assay for the zymotic acid is 9.72-32.98 ng/mL, and the detection limit is 5.79 ng/mL; in the analysis method of the dual-mode colorimetric fluorescence sensor, the linear detection range of the colorimetric method for detecting the mellea acid is 11.29-30.46 ng/mL, the detection limit is 8.45ng/mL, the visual detection accuracy is improved, the linear detection range of the fluorescence method for detecting the mellea acid is 8.49-46.19 ng/mL, the detection limit is 4.78ng/mL, and the combination of the two methods is independent response and mutually confirmed to show the excellent detection performance for the target object.

Description

Anti-mellea acid monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of food safety detection, in particular to a monoclonal antibody of anti-mellea acid and application thereof.

Background

Burkholderia gladioli pv. Cocovenens, BGC (rhizome of Calamus gladharia) easily pollutes cereal products (fermented corn flour, glutinous corn dumplings and the like), deteriorated tremella and produces secondary metabolite, namely, fermentation starter acid (Bongkrekekekie acid), which is a strong respiratory toxin, and the main target organs are liver, brain, kidney and the like, once poisoning is died due to respiratory failure, food poisoning events caused by the fermentation starter acid in China are more than dozens of times since 2014 according to incomplete statistics, and the mortality rate after eating by mistake is more than 50%.

For the detection of the fermentation broth acid, an instrumental analysis method needs expensive instruments, professional personnel for operation and maintenance and complex pretreatment, the chromatographic time is long, and the rapid detection on site cannot be realized. The immunoassay is an analysis technology based on the specific binding reaction of an antigen and an antibody, is suitable for field screening, is simple and rapid, has low cost, and is a common food safety rapid detection technology at present. InThe national patent already discloses a detection method of the zymotic acid in the food and the application thereof, and the sensitivity of the ELISA method is 50 mug/kg; and a detection method of the zymotic acid in the food and the application thereof, wherein the detection limit of the colloidal gold immunochromatography method is 5 mug/kg; the detection linear range of the enzyme-linked immunoassay method established by LiuxiuMei is 10³～10⁵Establishment of mu g/L (Liuxiumei, Wenwawa, Miyajie acid monoclonal antibody cell strain [ J]Sanitary research, 1996,025(004): 239-. For the prior art, high-sensitivity immunoassay for the rice ferment acid can not be achieved, and an immunoassay method with higher sensitivity is necessary along with frequent rice ferment acidosis events in recent years; in addition, the naked eye of the enzyme-linked immunoassay method has limited discrimination capability for the same color change depth, and the colloidal gold immunochromatography has false positive.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a high-sensitivity artificial antigen of a zymotic acid antibody and application thereof.

The first purpose of the invention is to provide a hybridoma cell strain secreting monoclonal antibody against the fermentation broth.

The second purpose of the invention is to provide a monoclonal antibody against the fermentation bacteria acid.

The third purpose of the invention is to provide the application of the hybridoma cell strain in establishing a detection method of the rice fermented lactobionic acid and/or preparing a detection kit of the rice fermented lactobionic acid.

The fourth purpose of the invention is to provide the application of the monoclonal antibody in establishing a method for detecting the fermentation broth and/or preparing a kit for detecting the fermentation broth.

The fifth purpose of the invention is to provide a method for detecting the fermentation broth.

The sixth purpose of the invention is to provide an immunological kit for detecting the mellea acid,

in order to achieve the purpose, the invention is realized by the following scheme:

the invention successfully prepares two artificial antigens by coupling the half antigen of the fermentation broth of which the structural formula is shown as the formula (I) to a carrier protein, prepares a hybridoma cell capable of secreting the antibody against the fermentation broth by immunizing animals with the complete antigen 1 of the fermentation broth of which the structural formula is shown as the formula (II-1), and further establishes the immunoassay method of the fermentation broth by taking the complete antigen 2 of the fermentation broth of which the structural formula is shown as the formula (II-2) as a coating antigen. The artificial antigen and the specific antibody prepared by the invention and the established immunoassay based on the artificial antigen and the specific antibody have high sensitivity and can supplement the requirement of low sensitivity at present. Wherein the linear detection range of the enzyme-linked immunosorbent assay for the zymotic acid is 9.72-32.98 ng/mL, and the detection limit is 5.79 ng/mL; in the analysis method of the dual-mode colorimetric fluorescence sensor, the linear detection range of the multicolor colorimetric method for detecting the mellea acid is 11.29-30.46 ng/mL, the detection limit is 8.45ng/mL, the visual detection accuracy is improved, the linear detection range of the fluorescence method for detecting the mellea acid is 8.49-46.19 ng/mL, the detection limit is 4.78ng/mL, and the combination of the two methods shows excellent detection performance for a target object due to independent response and mutual confirmation of the two methods. The dual mode is to mutually verify the accuracy of target object detection and avoid the accidental detection of the target object by one detection method. In addition, different target object concentrations in the multicolor colorimetric method can correspond to various rich color changes, so that the discrimination difference of naked eyes on single color is avoided, and the accuracy of visual detection is improved; the dual-mode fluorescence and multicolor colorimetric detection of the zymotic acid shows excellent detection performance on a target object due to independent response and mutual confirmation of the fluorescence method and the multicolor colorimetric method.

Therefore, the invention claims a hybridoma cell strain for secreting monoclonal antibody of fermentation broth, which is preserved in the China Center for Type Culture Collection (CCTCC) at 2021, 5, 12 months, and the preservation numbers are as follows: CCTCC No. C2021127.

The monoclonal antibody of anti-mellowoff's acid is secreted by the hybridoma cell strain.

The hybridoma cell strain is applied to establishing a detection method of the rice fermented mycolic acid and/or preparing a detection kit of the rice fermented mycolic acid.

The monoclonal antibody is applied to establishing a detection method of the fermentation broth and/or preparing a detection kit of the fermentation broth.

The invention also provides a preparation method of the gold nanoparticles, which comprises the following steps:

preparing a seed solution: and (3) fully mixing the sodium citrate solution and the chloroauric acid solution, wherein the molar ratio of the chloroauric acid to the sodium citrate is 1: 30-40 parts of;

preparing a growth solution; and (2) fully mixing the chloroauric acid solution, the nitrate solution and the ascorbic acid solution, wherein the molar ratio of the chloroauric acid to the nitrate to the ascorbic acid is 1: 1-2: 5-10;

mixing: and (3) fully mixing the prepared seed solution and the prepared growth solution, wherein the volume ratio is 1: and 10-15, changing the light yellow color of the solution into grayish blue to obtain the liquid.

Preferably, a seed solution is prepared: fully mixing the sodium citrate solution and the chloroauric acid solution, wherein the molar ratio of the chloroauric acid to the sodium citrate is 1: 30;

preparing a growth solution; and (2) fully mixing the chloroauric acid solution, the nitrate solution and the ascorbic acid solution, wherein the molar ratio of the chloroauric acid to the nitrate to the ascorbic acid is 1: 1.5: 5;

mixing: and (3) fully mixing the prepared seed solution and the prepared growth solution, wherein the volume ratio is 1: and 10, changing the light yellow color of the solution into grayish blue to obtain the product.

The gold nanoparticles are prepared by the preparation method.

And a preparation method of the fluorescent carbon dots, wherein a citric acid and urea mixed solution with N, N-dimethylformamide as a solvent reacts for 4-6 h at 160-200 ℃, wherein the mass ratio of the citric acid to the urea is 1: 2 to 4.

Preferably, the mixed solution of citric acid and urea with N, N-dimethylformamide as a solvent is reacted for 5 hours at 160 ℃, wherein the mass ratio of the citric acid to the urea is 1: 3.

The fluorescent carbon dot is prepared by the preparation method.

The invention also claims an immunological method for detecting the zymotic acid, and the monoclonal antibody is used as a detection antibody.

Preferably, the detection method is an indirect competition detection method for detecting the antigen, a compound shown as a structural formula (II) is used as a coating antigen,

more preferably, the detection method is an indirect competition detection method for detecting the antigen, a compound shown as a structural formula (II-2) is used as a coating antigen,

even more preferably, the method comprises the following steps:

the monoclonal antibody is used as a detection antibody;

coating a solid phase carrier with a compound shown as a structural formula (II-2) as a coating source;

washing and sealing the solid phase carrier;

adding the gradient-concentration standard solution of the zymotic acid and the detection antibody into the closed solid phase carrier, and incubating;

washing the solid phase carrier;

adding an enzyme-labeled secondary antibody into the washed solid phase carrier;

and (4) carrying out color development reaction by using TMB, and detecting a light absorption value.

Still further, more preferably, the method comprises the steps of:

1. coating: diluting the complete antigen 2 of the zymotic acid shown in the structural formula (II-2) as a coating antigen to 1 mu g/mL by using a pH 9.6 carbonic acid buffer solution, adding the diluted complete antigen into a hole of an enzyme-labeled plate, carrying out 100 mu L/hole, and carrying out full reaction at 37 ℃;

2. washing: pouring out the liquid in the holes, washing the plate for 2 times, and patting the liquid in the holes dry;

3. and (3) sealing: adding 200 μ L of sealing liquid into each hole, sealing at 37 deg.C for 2 hr, spin-drying the liquid in the hole, and inverted drying;

4. sample adding and incubation: the fermentation broth is diluted into a standard solution with a series of gradient concentrations, and the fermentation broth is diluted by 1.3 times from 100ng/mL in a gradient manner, wherein the total dilution is 12 concentrations (100ng/mL, 76.9ng/mL, 59.2ng/mL, 45.5ng/mL, 35.0ng/mL, 20.7ng/mL, 15.9ng/mL, 12.3ng/mL, 7.3ng/mL, 4.3ng/mL, 3.3ng/mL and 2.5ng/mL), 50 muL of the monoclonal antibody is added into each well, 10ng/mL is added, the reaction is fully performed at 37 ℃, the plate is washed for 5 times, and the liquid in the well is tapped;

5. adding a secondary antibody: adding 100 mu L of HRP-goat anti-mouse diluted by 5000 times into each hole, reacting in a water bath at 37 ℃ for 30min, washing the plate for 5 times, and patting the liquid in the holes dry;

6. color development: adding 100 μ L of TMB substrate solution into each well, and reacting at 37 deg.C for 10min

7. And (4) terminating: 50 μ L of 10% H by volume was added₂SO₄Terminating the display reaction;

8. and (3) determination: the absorbance at 450 nm of each well was measured using an enzyme linked immunosorbent assay.

Even more preferably, the method comprises the following steps:

the monoclonal antibody is used as a detection antibody;

washing and sealing the solid phase carrier;

washing the solid phase carrier;

adding a TMB substrate solution into a solid phase carrier to obtain a pre-color development system;

mixing the pre-color system with the fluorescent carbon dots, and detecting the fluorescence intensity of 620 nm;

pre-color system with 10% H₂SO₄Mixing to terminate the reaction, then mixing with CTAB, the gold nanoparticles and potassium iodide uniformly, and detecting the absorbance of 400-800 nm.

Still further, more preferably, the method comprises the steps of:

4. sample adding and incubation: the fermentation broth is diluted into a standard solution with a series of gradient concentrations, a 1.3-fold gradient is started from 1000ng/mL, a total of 12 concentrations (100ng/mL, 76.9ng/mL, 59.2ng/mL, 45.5ng/mL, 35.0ng/mL, 20.7ng/mL, 15.9ng/mL, 12.3ng/mL, 7.3ng/mL, 4.3ng/mL, 3.3ng/mL, 2.5ng/mL) are diluted, 50 muL of the monoclonal antibody is added into each well, 50 muL of the monoclonal antibody is added, the reaction is fully performed at 37 ℃, the plate is washed for 5 times, and the liquid in the well is tapped;

6. color development: adding 100 mu L of TMB substrate solution into each hole, and reacting at 37 ℃ for 10min to obtain a pre-color system;

7. taking 100 mu L of the obtained pre-color system, adding 50 mu L of the red fluorescent carbon dot solution, and measuring the fluorescence intensity of each hole at 620nm by using a measuring instrument at 540 nm;

8. taking 100 mu L of the pre-color system, and adding 50 mu L of H with the volume ratio of 10-15%₂SO₄Stopping the reaction by using a stop solution;

9. and (3) adding 50-100 mu L of 0.1-0.2M CTAB solution, 50-100 mu L of the gold nanoparticle solution and 50-100 mu L of 0.02-0.04M potassium iodide into the system for terminating the reaction in the step (8), fully reacting at room temperature, and detecting the absorbance of each hole at 400-800 nm.

The invention also claims an immunological kit for detecting the mellea acid, which contains the monoclonal antibody.

Preferably, the compound shown in the structural formula (II) is also contained as a coating source,

preferably, the compound shown as the structural formula (II-2) is also contained as a coating source,

more preferably, the composition also contains a stop solution H₂SO₄And TMB substrate solution.

More preferably, the gold nanoparticles and/or the fluorescent carbon dots are also contained.

Further preferably, the composition further comprises a CTAB solution and/or a potassium iodide solution.

Further preferably, the composition further comprises 0.1-0.2M CTAB solution and/or 0.02-0.04M potassium iodide solution.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a monoclonal antibody which is preserved in China center for type culture Collection in 2021, 5 months and 12 days, and the preservation numbers are as follows: the hybridoma cell with CCTCC No. C2021127 secretes, and the established immunoassay based on the hybridoma cell has high sensitivity and can supplement the requirement of low sensitivity at present. Wherein the linear detection range of the enzyme-linked immunosorbent assay for the zymotic acid is 9.72-32.98 ng/mL, and the detection limit is 5.79 ng/mL; in the analysis method of the dual-mode colorimetric fluorescence sensor, the linear detection range of the multicolor colorimetric method for detecting the mellea acid is 11.29-30.46 ng/mL, the detection limit is 8.45ng/mL, the visual detection accuracy is improved, the linear detection range of the fluorescence method for detecting the mellea acid is 8.49-46.19 ng/mL, the detection limit is 4.78ng/mL, and the combination of the two methods shows excellent detection performance for a target object due to independent response and mutual confirmation of the two methods.

Drawings

FIG. 1 is a graph showing the identification of the complete antigen 1 of meleagridic acid (as shown in formula (II-1)).

FIG. 2 is the identification chart of the complete antigen 2 of the fermentation broth (shown in formula (II-2)).

FIG. 3 is a graph of ELISA competition standards for a monoclonal antibody to mirinomycin acid.

FIG. 4 shows the morphology and color of gold nanoparticles with different aspect ratios.

FIG. 5 shows the morphology, particle size, excitation wavelength and emission wavelength of red fluorescent carbon dots.

FIG. 6 is a schematic diagram of a dual mode-colorimetric fluorescence sensor.

FIG. 7 is a graph showing the relationship between the wavelength and color of the inventive gold nanostars and the concentration of the fermentation broth.

FIG. 8 is a graph showing the change of fluorescence intensity of carbon dots prepared according to the present invention with the concentration of mirinogenic acid.

FIG. 9 is a standard curve graph of fluorescent and multicolor colorimetric dual-mode sensors for monoclonal antibodies to meleagranic acid.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 preparation of complete antigen of Miinomycelic acid

First, experiment method

1. Preparation of complete antigen 1 of Zymobacter oryzae acid (formula (II-1))

0.9mg of the mircotiana acid (the structural formula is shown as the formula (I)) is dissolved in 900 mu L of ethanesulfonic acid, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added under stirring, and the mixture is stirred at room temperature for 4 hours in a dark place to obtain an activated hapten which is called solution A. Wherein, the mol ratio of the half antigen of the zymotic acid (the structural formula is shown as formula I), the ethanesulfonic acid, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide is 1: 1: 1.5: 1.5,

10mg of bovine Lactoferrin (LF) was dissolved in a pH 9.6 carbonate buffer solution at a bovine lactoferrin concentration of 5mg/mL, referred to as solution B.

Dropwise adding the liquid A into the liquid B under ice bath stirring, wherein the molar ratio of the hapten in the liquid A to the carrier protein in the liquid B is 25: 1. after the dropwise addition, the pH was adjusted to 9.5 with 3M NaOH. Reacting overnight in a dark place, and dialyzing and purifying to obtain the complete antigen of the zymotic acid shown as the formula (II-1).

2. Preparation of complete antigen 2 of Zymobacter oryzae acid (formula (II-2))

10mg of Ovalbumin (OVA) was dissolved in a pH 9.6 carbonate buffer solution at a bovine lactoferrin concentration of 5mg/mL, referred to as solution C.

Dropwise adding the liquid A into the liquid C under ice bath stirring, wherein the molar ratio of the hapten in the liquid A to the carrier protein in the liquid C is 25: 1. after the dropwise addition, the pH was adjusted to 9.5 with 3M NaOH. Reacting overnight in the dark, and dialyzing and purifying to obtain the complete antigen of the zymotic acid shown as the formula (II-2).

Second, experimental results

The method comprises the steps of respectively scanning the light absorption of a hapten (the structural formula is shown as a formula (I)), a zymotic acid complete antigen 1 (the structural formula is shown as a formula (II-1)), a zymotic acid complete antigen 2 (the structural formula is shown as a formula (II-2)) and a carrier protein solution in an ultraviolet region (200-400 nm). As shown in figures 1 and 2, ultraviolet absorption characteristic peaks of a complete antigen 1 of the mircrobic acid (structural formula is shown as (II-1)) and a complete antigen 2 of the mircrobic acid (structural formula is shown as (II-2)) have a certain degree of red shift or blue shift relative to an original carrier protein, and the successful preparation of the complete antigen 1 of the mircrobic acid (structural formula is shown as (II-1)) and the complete antigen 2 of the mircrobic acid (structural formula is shown as (II-2)) is proved.

EXAMPLE 2 preparation of monoclonal antibody against Miinomycelic acid

First, experiment method

A6-8-week-old Balb/c mouse (Beijing Wintonlithan laboratory animal technology Co., Ltd.) is taken, the prepared 1mg/mL of the zymotic acid complete antigen 1 immune antigen shown as the formula (II-1) is mixed with Freund's complete adjuvant in equal amount, after complete emulsification, the mixture is injected into the abdomen and back of the mouse, and 100 mu L of the mixture is injected into each mouse. The first immunization adopts Freund complete adjuvant, the later booster immunization adopts Freund incomplete adjuvant, and the booster immunization is carried out once every 3 weeks for 4 times. Titers and inhibition were taken from tail vein one week after the third booster immunization. After the fourth boosting immunization, mice with higher titer and inhibition rate are selected for cell fusion, and the dose is doubled for boosting immunization once 3 days before the fusion.

Mouse myeloma SP2/0 cells were mixed with spleen cells at 5: 1, fusing under 50% PEG, washing, centrifuging, suspending with HAT medium, inoculating in 24-well culture plate, and culturing at 37 deg.C with 5% CO₂Half-exchange with HAT medium after 5 days in the incubator, and 10 days in which all the medium was changed to HT medium. When the cells in the plate grow to 1/3 of the culture hole area, the indirect ELISA method screens positive holes of the cells, and the zymotic acid complete antigen 2 shown in the structural formula (II-2) is used as a coating antigen during screening. Positive wells were further screened by indirect ELISA, limiting dilution cloning to approximately every well<1 cell, after 10 days, the cell strain obtained from the monoclonal well which is detected as positive and has better competition is the cell strain secreting the monoclonal antibody. After the hybridoma cells are subjected to expanded culture, the cell strains are collected and injected into the abdomen of a mouse, 1mL of the cell strains is injected into each mouse, and ascites of the mouse is taken 7 days later and purified to obtain the monoclonal antibody.

Second, experimental results

As shown in Table 1, the complete antigen 2 of fermentation broth represented by the structural formula (II-2) was used as a coating agent, the coating concentration was 1. mu.g/mL, and the drug concentration was 100 ng/mL.

Table 1 mouse antiserum characterization:

	potency of the drug	Suppression of
			No. 1 mouse	8K	71.2％
No. 2 mouse	16K	66.0％
			No. 3 mouse	16K	75.1％

Therefore, the monoclonal antibody obtained from the mouse No. 3 with the best titer and inhibition is selected to establish a detection method of a subsequent detection method, and the hybridoma cell strain secreting the monoclonal antibody is named as A5. Hybridoma cell line a5 was deposited in the chinese type culture collection, wuhan, 2021, 5 months and 12 days, with the deposit numbers: CCTCC No. C2021127.

Example 3 establishment of enzyme-linked immunoassay and specificity detection

First, experiment method

First, a standard curve was established using the monoclonal antibody prepared from mouse # 3 with the best medium titer and inhibition secreted by hybridoma cell line a5 of example 2, and the working concentration of the coating antigen was 1000ng/mL, and three sets of parallel experiments were performed (n-3).

The antiserum indirect competition detection method comprises the following steps of establishing a standard curve:

1. coating quilt

Diluting the complete antigen 2 of the zymotic acid shown in the structural formula (II-2) as a coating antigen to 1 mu g/mL by using a pH 9.6 carbonic acid buffer solution, adding the diluted complete antigen into a hole of an enzyme-labeled plate, keeping the diluted complete antigen at 100 mu L/hole, and standing overnight in a 37 ℃ water bath box;

2. washing machine

Pouring out the liquid in the hole, washing the plate for 2 times by a plate washing machine, and patting the liquid in the hole;

3. sealing of

Adding 200 μ L of sealing liquid into each hole, sealing at 37 deg.C for 2 hr, spin-drying the liquid in the hole, and placing in oven at 37 deg.C for 1 hr;

4. sample addition and incubation

The fermentation broth is diluted into a standard solution with a series of gradient concentrations, wherein the concentration of the fermentation broth is 1.3 times of that of the fermentation broth, the fermentation broth is diluted from 1000ng/mL, and a total of 12 concentrations (100ng/mL, 76.9ng/mL, 59.2ng/mL, 45.5ng/mL, 35.0ng/mL, 20.7ng/mL, 15.9ng/mL, 12.3ng/mL, 7.3ng/mL, 4.3ng/mL, 3.3ng/mL, 2.5ng/mL) are added into each well, 50 muL of a dilution of 10ng/mL antibody (monoclonal antibody secreted by hybridoma cell strain A5 in example 2) is added, after the reaction in a 37 ℃ water bath tank for 40min, a plate washing machine is used for 5 times, and the liquid in the wells is dry tapped;

5. adding a second antibody

Adding 100 mu L of HRP-goat anti-mouse diluted by 5000 times into each hole, reacting in a water bath tank at 37 ℃ for 30min, washing the plate for 5 times by a plate washing machine, and patting the liquid in the holes dry;

6. color development

Adding 100 μ L of TMB substrate solution into each well, and placing in a 37 deg.C water bath box for developing for 10 min;

7. terminate

50 μ L of 10% H by volume was added to each well₂SO₄A stop solution;

8. measurement of

Measuring the light absorption value of A450 nm of each hole by using an enzyme-linked immunosorbent assay detector;

9. computing

Calculating the IC10, IC20, IC50 and IC80 values of the inhibition curves by origin 8.5;

(II) specific detection

The detection method of the previous step is used for detecting the toxins commonly seen in the market so as to evaluate the specificity of the detection method.

The cross-reactivity R (%) is as follows:

r (%) ═ IC50 (mircotiana acid)/IC 50 (other common toxins) x 100%.

Second, experimental results

The standard curve is shown in FIG. 3. The IC50 value of the obtained enzyme-linked immunosorbent assay standard curve is 17.90ng/mL, the detection limit is (IC10)5.79ng/mL, and the linear detection range is 9.72-32.98 ng/mL.

The specificity detection is shown in table 2, and the results show that the prepared monoclonal antibody has better specificity and is not crossed with the common toxins on the market.

TABLE 2

Example 4 Synthesis of gold nanoparticles

First, experiment method

The seed growth induction method is adopted for synthesis, and the synthesis steps are as follows:

1. preparing seed liquid

And adding the sodium citrate solution into the chloroauric acid solution, mixing, heating to 80-100 ℃, and stirring for 3min, wherein the molar ratio of the chloroauric acid to the sodium citrate is 1: 30.

2. Preparing growth liquid

Mixing and stirring a chloroauric acid solution, a nitrate solution and an ascorbic acid solution for 2min, wherein the molar ratio of the chloroauric acid to the nitrate to the ascorbic acid is 1: 1.5: 5.

3. mixing

Adding the seed solution into the growth solution, wherein the volume ratio is 1: and 10, mixing and stirring for 2-3 min, and changing the solution from light yellow to grey blue to obtain the gold nanoparticles.

Second, experimental results

Obtaining the gold nanoparticles shown in FIG. 4, wherein the gold nanoparticles with different particle diameters have different shapes and colors under the same proportion: blue gold nanoparticles are similar to star shapes, and have more corners around the gold nanoparticles and larger particle sizes; the corner of the purple gold nano particles becomes short and the particle size is intermediate; the red gold nanoparticles were substantially rounded and had the smallest particle diameter.

EXAMPLE 5 Synthesis of Red fluorescent carbon dots

First, experiment method

The compound is synthesized by a one-step hydrothermal method, and the synthesis steps are as follows:

1. dissolving citric acid and urea by using 10mL of N, N-dimethylformamide, wherein the dosage of the citric acid is 1-2 g, the dosage of the urea is 3-6 g correspondingly, and the mass ratio of the citric acid to the urea is 1: 3.

2. Transferring the mixed solution in the last step to a reaction kettle, and heating for 5 hours at 160 ℃.

Second, experimental results

The carbon dots as shown in FIG. 5 were prepared. From fig. 5A, it can be seen that the carbon dots are uniformly dispersed, and fig. 5B is a high resolution electron micrograph of one of the carbon dots of fig. 5A, the lattice fringes are clearly visible, the lattice spacing is 0.21nm, fig. 5C is a carbon dot particle size distribution between 1.75nm and 4.25nm, from fig. 5D, it can be seen that the excitation wavelength (Ex) of the carbon dots is 540nm, and the emission wavelength (Em) is 620 nm.

EXAMPLE 6 method of detecting Mimejic acid with fluorescent and multicolor colorimetric Dual-mode sensor

FIG. 6 shows a schematic diagram of a fluorescent and multicolor colorimetric dual-mode assay for detecting mellitic acid, in which the amount of the sample antigen, namely, mellitic acid, to be detected by the detection system in an indirect competitive assay method affects the substrate TMB⁺The amount of (a) further:

for the fluorescence method: substrate TMB⁺Has an ultraviolet absorption peak at 650nm, coincides with the fluorescence emission spectrum of Carbon Dots (CDs), and is based on the internal filtering effect⁺Can absorb the energy of the emission wavelength of CDs, and specifically comprises the following components: the amount of the zymotic acid in the sample to be detected of the detection system is TMB⁺The amount of (c) is inversely proportional, and the fluorescence intensity of CDs is dependent on TMB⁺Is increased and decreased, so that the content of the fermentation broth can be measured by measuring the fluorescence intensity of CDsAnd (4) quantifying.

For in multicolor colorimetry: blue color TMB⁺Due to the reaction with 10% H₂SO₄Oxidation reaction occurs to become yellow TMB²⁺Further, yellow TMB²⁺And nano-star to perform oxidation reduction reaction (TMB) in the presence of surfactant CTAB²⁺+2Au＝TMB+2Au⁺) The particle size of the nano-gold star is continuously reduced, and the colors of the nano-gold star with different particle sizes are different under visible light, so that the solution color of the system to be detected is changed. Specifically, the method comprises the following steps: the amount of the zymotic acid in the sample to be detected of the detection system is TMB⁺Is inversely proportional to the amount of TMB⁺Is determining the amount of TMB²⁺The particle size of the nano-gold star is determined by TMB²⁺The amount of the enzyme increases and becomes short, so that the content of the fermentation broth can be quantified by measuring the absorbance of the system to be measured.

First, experiment method

A standard curve was established using the monoclonal antibody prepared from mouse # 3 having the best neutralizing potency and inhibition secreted by hybridoma cell line A5 of example 2, and the coating concentration of the coating antigen (kojic acid complete antigen 2 represented by structural formula (II-2)) was 1000ng/mL, and three sets of parallel experiments were performed (n-3).

The detection steps are as follows:

1. 1-5, antiserum indirect competitive detection step by the enzyme-linked immunosorbent assay of the same embodiment 3;

6. color development: adding 200 μ L of TMB substrate solution into each well, and placing in a 37 deg.C water bath box for developing for 10 min;

7. taking 100 mu L of the solution developed in the step 6, adding 50 mu L of the 10M red fluorescent carbon dot solution prepared in the example 5, and measuring the fluorescence intensity of each well at 620nm by using an enzyme-linked immunosorbent assay detector at 540 nm;

8. taking 100 mu L of the solution after the color development in the step 6, adding 50 mu L of 10% H₂SO₄Stopping the reaction by using a stop solution;

9. adding the system for terminating the reaction in the step 8 into a solution containing 50 mu L of 0.1-0.2M CTAB solution, 50 mu L of 50mM gold nanoparticle solution prepared in the embodiment 4 and 50 mu L of 0.02M potassium iodide, reacting for 10min, and measuring the absorbance of each hole at 400-800 nm by using an enzyme linked immunosorbent assay instrument.

Second, experimental results

FIG. 7 shows the relationship between the fluorescence intensity of carbon spots and the concentration of the fermentation broth; the wavelength and color of gold nanoparticles as a function of the concentration of the mircolebic acid are shown in fig. 8.

The standard curve is shown in FIG. 9. Wherein the detection limit of the multicolor colorimetric method is 8.45ng/mL, and the linear detection range is 11.29-30.46 ng/mL; the detection limit of the fluorescence method is 4.78ng/mL, and the linear detection range is 8.49-46.19 ng/mL.

EXAMPLE 7 kit for detecting Mimejic acid by fluorescent and multicolor colorimetric dual-mode sensor

A, make up

Anti-mirinogenic acid monoclonal antibody: as the culture is preserved in China center for type culture Collection in 2021, 5 months and 12 days, the preservation numbers are as follows: secreting a hybridoma cell strain with CCTCC No. C2021127;

coating source: the structural formula of the compound is shown as (II-2),

stop solution (volume fraction of 10% H₂SO₄) Gold nanoparticles, fluorescent carbon dots, a CTAB solution, and a potassium iodide solution;

wherein the gold nanoparticles were prepared according to the method of example 4, and the fluorescent carbon dots were prepared according to the method of example 5.

Second, use method

The same as in example 6.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A hybridoma cell strain secreting monoclonal antibodies against zymotic acid is characterized in that the hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 12 days, and the preservation numbers are as follows: CCTCC No. C2021127.

2. An anti-mircromallic acid monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The hybridoma cell strain of claim 1, wherein the hybridoma cell strain is used for establishing a method for detecting the presence of the enzyme-producing bacterium and/or preparing a kit for detecting the presence of the enzyme-producing bacterium.

4. The use of the monoclonal antibody of claim 2 in establishing a method for detecting mirinogenic acid and/or in preparing a kit for detecting mirinogenic acid.

5. A method for detecting a meleuoff acid, which comprises using the monoclonal antibody according to claim 2 as a detection antibody.

6. The detection method according to claim 5, wherein the detection method is an indirect competitive detection method for detecting an antigen, and comprises using a compound represented by the formula (II) as a coating antigen,

7. the detection method according to claim 5, wherein the detection method is an indirect competitive detection method for detecting an antigen, and comprises using a compound represented by the formula (II-2) as a coating antigen,

8. an immunological kit for detecting mellea acid, comprising the monoclonal antibody of claim 2.

9. The immunological kit as claimed in claim 8, further comprising a compound represented by the structural formula (II) as a coating antigen,

10. the immunological kit as claimed in claim 8, further comprising a compound represented by the structural formula (II-2) as a coating antigen,