CN111007245A

CN111007245A - Quick detection kit for flow lag immune time resolution fluorescence of ribes diacetylenium sickle enol

Info

Publication number: CN111007245A
Application number: CN201911122490.1A
Authority: CN
Inventors: 张奇; 唐晓倩; 白艺珍; 张文; 李培武
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-04-14
Anticipated expiration: 2039-11-15
Also published as: CN111007245B

Abstract

The invention relates to a quick detection kit for flow lag immune time resolution fluorescence of sickle knife enol of ribes diacetylenii. It includes fluorescence test paper strip and contains the sample reaction bottle of the anti diacetyl grass fusarium enol monoclonal antibody freeze-dried product of europium mark, wherein: fluorescence test paper strip include the welt, the one side from the top down of welt pastes water absorption pad, detection pad and sample pad in proper order, and adjacent each pad is in junction overlap connection, detect and fill up and use the nitrocellulose membrane as the base pad, top-down transversely sets up quality control line and detection line on the nitrocellulose membrane, the quality control line peridium has rabbit anti mouse polyclonal antibody, peridium is diacetyl grass fusarium enol complete antigen (DAS-OVA) on the detection line. Can accurately and quickly determine the content of the diacetyl ribes sickle knife enol.

Description

Quick detection kit for flow lag immune time resolution fluorescence of ribes diacetylenium sickle enol

Technical Field

The invention belongs to the field of mycotoxin detection, and particularly relates to a quick fluorescence detection kit for flow lag time resolution of sickle knife enol of ribes diacetylenium.

Background

Mycotoxins (mycotoxins) are toxic secondary metabolites produced by fungi during their growth, are generally low molecular substances, and most mycotoxins are heat stable substances. Since the discovery of mycotoxins, over 300 mycotoxins have been identified, wherein fusarium toxin has the characteristics of strong toxicity and high pollution frequency, and trichothecene compounds are a kind of mycotoxin with higher pollution level and greater harm. Trichothecenes are divided into two types A and B, and the ribes diacetylenium sickle enol belongs to the trichothecene A type. Diketofusarenol has strong acute toxicity and LD (LD) to rats₅₀0.75mg/kg, and has strong thermal stability, so that the toxin can not be destroyed by common cooking means. Post-intoxication clinical manifestations of severe dermatitis, nausea, vomiting, bloody diarrhea, damage to the bone marrow hematopoietic system, nervous system disturbances, anorexia and death. In view of its harmful effects and its widespread occurrence in food and feed, the contents are strictly limited in countries around the world. With the continuous improvement of living standard, the requirements of people on food quality safety are higher and higher. In order to improve the food quality safety level in China and meet the safety consumption requirements of people to a greater extent, the monitoring on the sickle knife enol of ribes diacetylenium in grains is enhanced, and an accurate and efficient detection technology is developed.

The existing detection method of mycotoxin mainly comprises an instrumental analysis method and an immunoassay method. The instrumental analysis methods such as high performance liquid chromatography, liquid chromatography-mass spectrometry combined method and the like have high sensitivity and good accuracy, but the instruments are expensive, the sample pretreatment process is complicated, the consumed time is long, the requirements on the experimental environment are high, and the like, so that the rapid detection is difficult to realize. The immunoassay method is an analysis method based on the specific recognition effect and reversible binding reaction of an antigen and an antibody, has high selectivity and sensitivity, greatly simplifies and shortens the sample processing time compared with an instrumental analysis method, and saves the detection cost. The time-resolved fluoroimmunoassay (TRFICA) utilizes europium as a high-affinity probe, has the advantages of high sensitivity, stable property, avoidance of interference of fluorescence background, short detection time and the like, and is very suitable for developing a rapid detection method for pesticide residues.

Therefore, the development of the quick detection kit for the flow lag immune time-resolved fluorescence of the sickle enol of ribes diacetylenii has great necessity and very important significance.

Disclosure of Invention

The invention aims to solve the problem of providing a quick detection kit for the flow delay of sickle knife enol of diacetyl ribes for immune time resolution fluorescence.

A quick detection kit for flow lag immunity time resolution fluorescence of sickle knife enol of diacetyl is disclosed, which comprises a fluorescent test strip and a sample reaction bottle containing europium-labeled monoclonal antibody freeze-dried product of anti-sickle knife enol of diacetyl, wherein: the fluorescent test strip comprises a lining plate, wherein a water absorption pad, a detection pad and a sample pad are sequentially adhered to one surface of the lining plate from top to bottom, the adjacent pads are connected in an overlapping manner at the joint, the detection pad takes a nitrocellulose membrane as a base pad, a quality control line and a detection line are transversely arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the detection line is coated with a diacetyl ribes fusarenol complete antigen (DAS-OVA); the anti-diacetylebergens fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO. C201881. The hybridoma cell strain DAS5G11E7 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 and 3, and the preservation address is China, Wuhan and Wuhan university with the preservation number of CCTCC NO. C201881.

According to the scheme, the europium-labeled anti-diacetylebergene fusarenol monoclonal antibody is prepared according to the following method: dialyzing the anti-diacetyl sickle knife enol monoclonal antibody with a carbonate buffer solution, fully and uniformly mixing the dialyzed anti-diacetyl sickle knife enol monoclonal antibody with an europium-labeled reagent according to the mass ratio of 0.5-2: 1, standing overnight, separating the europium-labeled anti-diacetyl sickle knife enol monoclonal antibody through a Sephadex G-50 chromatographic column, eluting, and collecting a target product.

According to the scheme, the water absorption pad in the fluorescent test strip is 15-20 mm long and 3-4 mm wide; the detection pad is 25-30 mm long and 3-4 mm wide; the sample pad is 12-18 mm long and 3-4 mm wide, and the overlapping length of each adjacent pad is 1-3 mm.

According to the scheme, the distance between the detection line on the detection pad in the fluorescent test strip and the upper edge of the nitrocellulose membrane is 15-20 mm, and the distance between the quality control line and the detection line is 5-10 mm; the sample reaction bottle is a 1-5 mL bayonet bottle.

According to the scheme, 480-1000 ng of complete antigen coating amount of diacetyl ribes sickle knife enol required by each centimeter detection line on a detection pad in the fluorescent test strip; coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter of quality control line is 100-900 ng; the content of europium-labeled anti-diacetyl ribes fusarium enol monoclonal antibody freeze-dried product in the sample reaction bottle is 0.2-1.0 ug.

According to the scheme, the fluorescent test strip is obtained by adopting the following method:

(1) cutting the absorbent paper into absorbent pads with required sizes;

(2) preparation of a detection pad:

preparing a DAS-OVA antigen coating solution with the concentration of 0.125-0.8 mg/mL from a sickle enol complete antigen (DAS-OVA), transversely coating the DAS-OVA antigen coating solution on a nitrocellulose membrane in a membrane scratching mode to obtain a detection line, and drying the detection line for 30-60 minutes at 37-40 ℃; preparing a coating solution with the concentration of 0.1-1.0 mg/mL by using a rabbit anti-mouse polyclonal antibody, transversely coating the coating solution on a nitrocellulose membrane in a membrane scratching mode to obtain a quality control line, and drying the nitrocellulose membrane for 30-60 minutes at 37-40 ℃;

(3) preparation of sample pad:

putting the glass fiber membrane into the sealing liquid for completely soaking, taking out the glass fiber membrane, drying the glass fiber membrane for 3 to 6 hours at the temperature of between 37 and 40 ℃ to obtain a required sample pad, and storing the sample pad in a dryer at room temperature;

(4) assembling the fluorescent test strip:

and sequentially sticking the water absorption pad, the detection pad and the sample pad on one surface of the lining plate, and overlapping and connecting adjacent pads at the joint to obtain the fluorescent test strip.

According to the scheme, the coating buffer solution used for preparing the coating solution of the sickle knife enol complete antigen (DAS-OVA) of the ribes diacetylenii in the preparation of the fluorescent test strip is as follows: every 10mL contains bovine serum albumin 0.1g, sodium chloride 0.08g, potassium chloride 0.002g, sodium azide 0.002g, disodium hydrogen phosphate dodecahydrate 0.029g, and potassium dihydrogen phosphate 0.002 g;

the coating buffer used in the preparation of the rabbit anti-mouse polyclonal antibody coating solution is as follows: each 10mL of the solution contains 0.08g of sodium chloride, 0.002g of potassium chloride, 0.002g of sodium azide, 0.029g of disodium hydrogen phosphate dodecahydrate and 0.002g of potassium dihydrogen phosphate;

the confining liquid used in the preparation of the fluorescent test strip is as follows: each 100mL of the composition contains 0.5-2 g of egg white albumin, 1-2 g of sucrose, 0.8g of sodium chloride, 0.02g of potassium chloride, 0.02g of sodium azide, 0.29g of disodium hydrogen phosphate dodecahydrate and 0.02g of potassium dihydrogen phosphate.

According to the scheme, the quick detection kit for the flow lag immunization time-resolved fluorescence of the sickle knife enol of diacetyl UE also comprises a sample diluent and a sample diluent straw, wherein the sample diluent is a Tween 20 aqueous solution with the volume fraction of 0.01-0.30%.

The application of the quick detection kit for flow lag immune time resolution fluorescence of the sickle enol of ribes diacetylenii in the detection of the content of the sickle enol of ribes diacetylenii comprises the following steps: adding a sample solution to be detected into a sample reaction bottle, uniformly mixing, inserting a fluorescent test strip, reacting at 37 ℃ for 6-10 minutes, and detecting by using a time-resolved fluorescence tester to obtain the ratio of the fluorescence intensity value of a detection line (T) on the fluorescent test strip to the fluorescence intensity value of a quality control line (C); based on a relation curve between the ratio (T/C) of fluorescence intensity of a fluorescence test strip detection line to fluorescence intensity of a quality control line and concentration of the diacetyl ribes sickle knife enol, the content of the diacetyl ribes sickle knife enol in the sample liquid to be detected is obtained.

According to the scheme, the relation curve of the ratio (T/C) of the fluorescence intensity of the detection line of the fluorescent test strip to the fluorescence intensity of the quality control line and the concentration of the sickle knife enol of the ribes diacetylenii is obtained by adopting the following method:

(1) preparing a series of concentration gradient solutions of a sickle knife enol standard substance of ribes diacetylenii;

(2) adding a proper amount of the sickle knife enol standard substance solution with each concentration into a sample reaction bottle respectively, mixing uniformly, inserting a fluorescent test strip, reacting for 6 minutes at 37 ℃, and detecting by a time-resolved fluorescence immunoassay analyzer to obtain time-resolved fluorescence intensity values of a detection line (T) and a quality control line (C) on each fluorescent test strip, so as to obtain the ratio (T/C) of the fluorescence intensity of each fluorescent test strip detection line to the fluorescence intensity of the quality control line;

(3) and obtaining a relation curve of the ratio (T/C) of the fluorescence intensity of the detection line of the fluorescent test strip to the fluorescence intensity of the quality control line and the concentration of the sickle knife enol of the ribes diacetylenii by fitting.

This research couples diacetyl grass fusarium enol monoclonal antibody and rare earth element europium label reagent mutually, diacetyl grass fusarium enol time resolution fluorescence immunochromatography test paper strip of high sensitivity has been developed, utilize the time resolution fluorimeter to carry out the ration, time resolution fluorescence immunochromatography method has been established, it is high to have sensitivity, stable in nature, avoid the interference of fluorescence background, advantages such as detection time is short (general detection only needs 6-8min), very be fit for developing the pesticide residue short-term test method, can quick sensitive detection diacetyl grass fusarium enol.

The invention has the beneficial effects that:

the rapid flow hysteresis immunoassay time-resolved fluorescence test kit for the sickle knife enol of ribes diacetylenium provided by the invention can accurately and rapidly test the content of the sickle knife enol of ribes diacetylenium. The sample detection limit of the sample on the diacetyl sickle knife enol is 0.1ng/mL (11 times of blank sample detection, the SD value is calculated, and the detection limit is 3 SD). The test strip is applied to detection of an actual sample, the detection result is compared with an HPLC method, the recovery rate is 87.7% -115.3%, the result accords with the correlation coefficient and reaches 0.988(R2), and the test strip can be used for detection of a practical sample of the sickle enol of ribes diacetylenii.

Drawings

FIG. 1 is a schematic structural diagram of a fluorescent test strip in a fast fluorescence detection kit for immunoassay time resolution based on flow delay of sickle knife enol of ribes diacetylenium provided by the present invention. In the figure: 1 sample pad, 2 detection lines, 3 quality control lines and 4 water absorption pads.

Fig. 2 is affinity measurement data of a sickle enol monoclonal antibody based on ribes diacetate provided by the present invention;

fig. 3(a) shows the cross reaction result between the ribes diacetate fusarenol monoclonal antibody and other mycotoxins provided by the present invention; (b) the invention provides a standard curve of a ribes diacetate fusarenol enzyme-linked immunosorbent assay method established by the ribes diacetate fusarenol monoclonal antibody.

Detailed Description

Obtaining of anti-diacetyl ribes fusarenol monoclonal antibody

The anti-diacetyl ribes fusarium enol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO. C201881, and the preparation method comprises the following steps:

injecting a hybridoma cell strain DAS5G11E7 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the concrete operations are as follows: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. Centrifuging at 12000r/min at 4 deg.C for more than 30min, discarding precipitate, filtering the obtained supernatant with double-layer filter paper, 1/10 adding phosphate buffer solution with molarity of 0.1mol/L and pH of 7.4, adjusting pH of the mixture to 7.4 with 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in ice bath to final concentration of 0.277g/mL, standing at 4 deg.C for more than 2 hr, then 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate with phosphate buffer solution with the molar concentration of 0.01mol/L, pH of 7.4 of the volume of original ascites volume 1/10, filling into a dialysis bag, dialyzing for two days with 0.01mol/LPBS, dialyzing for two days with PB again, taking out the protein solution in the dialysis bag, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing at-70 ℃, and freeze-drying in a freeze-dryer. Collecting freeze-dried powder, namely the purified ribes diacetate resistant sickle enol monoclonal antibody;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water into 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate.

The subtype of the ribes diacetate resisting sickle enol monoclonal antibody secreted by the hybridoma cell strain DAS5G11E7 is identified to be IgG2b by using a commercial subtype identification kit.

The titer of the antibody obtained by ascites purification of the mice can reach 3.2 multiplied by 10 measured by the conventional non-competitive enzyme-linked immunosorbent assay (ELISA)⁵I.e. 3.2X 10 dilution of antibody⁵The solution test result is positive. The sensitivity of the method to the sickle enol diacetate is 3.08ng/mL by using the conventional indirect competition ELISA. The cross-reactions with other mycotoxins, T2 toxin, HT2 toxin, vomitoxin, 3-acetyl deoxynivalenol, ochratoxin, fumonisin were all less than 0.01% (Table 1; FIG. 3). The specificity of the antibody can be evaluated by the cross-reactivity. Detecting DAS5G11E7 monoclonal antibody by indirect competition ELISA method, and detecting DAS, T2 toxin, HT2 toxin, DON, 3-ACDON, OTA and FB₁Preparing standard solutions with series concentrations, respectively adding the standard solutions and an antibody with the same volume into an ELISA plate, incubating for 1h, and performing other steps with an indirect competitive ELISA method. And (3) drawing a competitive inhibition curve by taking the concentration of the toxin standard substance as an abscissa and an OD value B/B0 measured by an enzyme-labeling instrument at 450nm as an ordinate, and determining the cross reaction rate by calculating the ratio of the IC50 values of the DAS to other toxins. The calculation formula is as follows:

CR% (IC50DAS/IC50 other toxins) × 100.

Table 1 cross-reactivity of DAS5G11E7 with other toxins.

The affinity of DAS5G11E7 was determined using an indirect non-competitive ELISA. Coating an enzyme label plate with DAS-OVA according to the concentration of 1.0, 0.5, 0.25 and 0.125 mu g/mL, 100 mu L/hole, 37 ℃ and 2 h; after blocking with blocking solution for 1h, the antibody (dilution factor 1:2) diluted with PBS was added to the ELISA plate, and the rest steps were performed in the same manner as in the indirect non-competitive ELISA method. With the measured OD450 values as ordinate and the logarithm of the antibody concentration (mol/L) as abscissa, 4S-shaped curves of 4 concentrations were made. The maximum OD value at the top of each S-curve, i.e., ODmax, was found, and the antibody concentration corresponding to 50% ODmax of each curve was found. Any two of the 4 concentrations are combined into one group according to the formula Ka ═ n-1)/2(n [ Ab']t-[Ab]t) calculating the affinity constant of the antibody, wherein [ Ab']t、[Ab]t is the antibody concentration corresponding to the two 50% maximum OD values in each group, n is the multiple of the envelope antigen concentration in each group (including three ratios of 1:2, 1: 4, 1: 8), and 6 Ka values are obtained. The six obtained Ka values are averaged to obtain the anti-diacetyl-grass fusarenol mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method with the affinity of 5.4 multiplied by 10⁸L/moL (FIG. 2).

Screening of hybridoma cell line DAS5G11E7

1. Animal immunization

A BALB/c mouse of 6-7 weeks old is immunized by using a sickle knife enol complete antigen DAS-BSA prepared in a laboratory. The first immunization is that after the diacetyl sickle enol complete antigen and equivalent volume of Freund's complete adjuvant are emulsified, subcutaneous multi-point injection is carried out on the back of the neck of a mouse. The second immunization is carried out after 4 weeks, and the second immunization is emulsified by a Freund incomplete adjuvant and isovolumetric sickle enol complete antigen and injected into the abdominal cavity of a mouse. The third immunization was carried out at 4 weeks intervals from the second immunization in the same manner as the first immunization, and the fourth immunization was carried out 3 weeks after the third immunization in the same manner as the second immunization, which was also administered intraperitoneally. The 4 immunization doses were identical and were 70. mu.g per mouse. And (3) collecting blood from tail veins 8-10 days after each immunization of the first 3 times, separating serum, and detecting the serum titer of the mice by adopting indirect ELISA. 8 days after 3 rd immunization, blood is collected by breaking the tail, and mice corresponding to serum with relatively high titer and sensitivity are selected for carrying out the last boosting immunization, wherein the immunization dose is 2 times of that of the mice.

2. Cell fusion

After 3 days of boosting immunity, adopting PEG with 50% of polyethylene glycol and molecular weight of 1450 as fusion agent, and making cell fusion according to conventional method, and its concrete steps are: killing the mouse by removing neck under aseptic condition, taking out spleen, crushing spleen with homogenizer, separating spleen cells with filter screen, mixing with mouse myeloma cell SP2/0 at a ratio of 5: 1, centrifuging, resuspending mixed cells with RPMI-1640 basic culture solution, centrifuging, and discarding supernatant. Adding 1-2mL of 50% PEG, adding 10-20mL of RPMI-1640 basic culture solution in an adherent manner for 1 minute in a common use, centrifuging, discarding the supernatant, re-suspending the fused cells at the bottom of the tube by using 20mL of cell complete culture medium containing 1% HAT, adding the suspended cells into 80mL of semisolid culture medium, uniformly mixing, adding the mixture onto a 6-well cell culture plate, culturing at 1.5 mL/well, and placing the mixture in a carbon dioxide incubator at 37 ℃. The complete cell culture medium containing 1% HAT contains 20% (volume percent) of fetal calf serum, 75% (volume percent) of RPMI-1640 basic culture solution, 1% (weight percent) of L-glutamine, 1% (volume percent) of HEPES, 1% (volume percent) of diabody (10000 units per milliliter of penicillin and 10000 micrograms per milliliter of streptomycin), 2% (volume percent) of growth factor (HFCS) and 1% (weight percent) of hypoxanthine-aminopterine-thymidine, namely HAT and methylcellulose, which are purchased from sigma-Aldrich company.

Screening and cloning of cell lines

And (3) picking out the clone from the culture medium by using a micropipette when the cell colony grows to be visible by naked eyes 2-3 weeks after the cells are fused, transferring the clone to a 96-hole cell culture plate, culturing by using HAT liquid, and sucking culture supernatant for detection when the cells grow to the bottom of 2/3 holes. Adopting a two-step screening method, wherein in the first step, an indirect ELISA method is adopted to screen out positive holes which resist sickle knife enol diacetate but not resist carrier protein BSA; in the second step, an indirect competition ELISA method is adopted to detect the positive holes screened in the first step, sickle enol diacetate is used as a competition source, and holes (light absorption values) with higher light absorption values and higher sensitivity are selectedHigher means that the final measurement value of the well in which the competitive antigen is 0, that is, the positive control well, is higher, and higher sensitivity means that the competitive antigen concentration is IC even when the inhibition rate is 50%₅₀Smaller value), carrying out subcloning by using a limiting dilution method, carrying out detection by using the same two-step method after subcloning, and repeating the subcloning for 4-5 times to obtain a hybridoma cell strain DAS5G11E 7. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 months and 3 days, wherein the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO: C201881.

Determination of variable region sequence of anti-ribes diacetate fusarenol monoclonal antibody hybridoma cell strain DAS5G11E7 antibody

(1) Extracting total RNA: extracting total RNA capable of generating hybridoma cell strains DAS5G11E7 by adopting a total RNA extraction kit of Tiangen company according to an instruction;

(2) synthesis of cDNA: taking the total RNA obtained in the step 1 as a template, and oligo (dT)15 as a primer according to SuperScript^TM-2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT)15 was purchased from Invitrogen;

(3) the PCR method is to design primers according to conserved sites of a mouse antibody gene sequence in GENBANK, amplify heavy chain and light chain variable region genes of the antibody by using CDNA as a template, wherein the PCR program comprises the steps of amplifying for 30 cycles at 94 ℃ for 30S at 58 ℃ for 45S at 72 ℃ for 1min, finally extending for 10min at 72 ℃, separating a PCR product by agarose gel electrophoresis at 1% (weight percentage), purifying and recovering DNA fragments by using a kit, connecting the DNA fragments to a vector pMD18-T, transforming escherichia coli DH5 α competent cells, picking up positive clones, and sending the positive clones to Hippocampus Moriniensis Biotech limited for sequencing, wherein the sequences of the primers are respectively a heavy chain variable region primer of 5 '-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and a 5'-CAG GGG CCA GTGGAT AGA CAG ATG GGG G-3' (28mer), wherein S, M, R and W are merged bases, M ═ A/C, R ═ A/G, S ═ G/C, W ═ A/T, and a light chain variable region primer of 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCGTTT TAT TTC CAG CTT GGT CCC-3' (24 mer).

Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 351bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 117 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence is 324bp long and is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 108 amino acids, and the sequence is shown as SEQ ID NO. 4.

Example 1: diacetyl ribes sickle knife bacterium enol immune time-resolved fluorescence rapid detection kit and application thereof

The rapid detection kit for the flow lag immunity time-resolved fluorescence of the sickle enol of diacetyl grass comprises a fluorescent test strip, a sample reaction bottle (containing europium-labeled monoclonal antibody freeze-dried product of the sickle enol of diacetyl grass), a sample diluent and a sample diluent straw. The fluorescent test strip comprises a paperboard, one surface of the paperboard is sequentially pasted with a water absorption pad, a detection pad and a sample pad from top to bottom, the adjacent pads are connected in an overlapped mode at the joint, the overlapped length is 1-2mm, and the fluorescent test strip comprises: the absorbent pad is 15mm long and 4mm wide; the detection pad is 25mm long and 4mm wide; the sample pad was 13mm long and 4mm wide. The detection pad uses the nitrocellulose membrane as a base pad, a quality control line and a detection line are transversely arranged from top to bottom on the nitrocellulose membrane, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, the detection line is coated with a diacetyl grass fusarium enol complete antigen, the distance between the detection line and the upper edge of the nitrocellulose membrane is 15mm, and the distance between the quality control line and the detection line is 5 mm.

Obtaining the fluorescent test strip:

(1) preparation of absorbent pad

Cutting the absorbent paper into pieces with the length of 15mm and the width of 4mm to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

preparing 0.8mg/mL coating solution from a ribes diacetylenium fusarenol complete antigen (DAS-OVA) by using the coating buffer solution; transversely coating the nitrocellulose membrane at a position 15mm away from the nitrocellulose membrane by a wire spraying mode to obtain a detection wire, wherein the coating amount of DAS-OVA required by each centimeter of the detection wire is 480ng, and then drying for 30 minutes at 37 ℃;

the coating buffer solution is as follows: 0.1g of bovine serum albumin, 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride and 0.002g of potassium dihydrogen phosphate, and adding water to a constant volume of 10 mL;

coating of quality control line:

coating buffer solution is used for preparing a rabbit anti-mouse polyclonal antibody into coating solution with the concentration of 0.25 mg/mL; transversely coating the test line on a nitrocellulose membrane at a position 6mm away from the test line in a line spraying manner to obtain a quality control line, wherein the coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter of the quality control line is 100ng, and then drying for 1 hour at 37 ℃;

the coating buffer solution is prepared by adding water into 0.002g of sodium azide, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride and 0.002g of potassium dihydrogen phosphate to a constant volume of 10 mL;

(3) preparation of sample pad:

cutting the glass fiber membrane into pieces with the length of 13mm and the width of 4mm, soaking in a sealing solution, taking out, drying at 37 ℃ for 6 hours to obtain a sample pad, and then placing in a dryer for storage at room temperature;

the confining liquid is prepared by adding water into 1g of ovalbumin, 2g of cane sugar, 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate to achieve a constant volume of 100 mL;

(4) assembling the fluorescent test strip:

and (3) sequentially sticking a water absorption pad, a detection pad, a fluorescence labeling antibody reaction pad and a sample pad on one surface of the paperboard from top to bottom, and overlapping and connecting adjacent pads at the joint, wherein the overlapping length is 2mm, so that the fluorescence test strip (shown in figure 2) is obtained.

Obtaining the europium-labeled anti-diacetyl grass fusarenol monoclonal antibody: 1mg of the monoclonal antibody was washed with 100mmol/L carbonate buffer pH9.3 repeatedly for 6 times, and then mixed well with 2mg of europium-labeled reagent at 4 ℃ overnight. Then adding the europium-labeled anti-diacetyl sickle enol monoclonal antibody into a Sephadex G-50 chromatographic column of 1.9cm multiplied by 60cm, eluting with 50mmol/L Tris-HCl eluent containing 0.9% NaCl, collecting effluent liquid (1 ml/tube), measuring an absorbance value (A280nm) tube by tube, and combining peak tubes to obtain the target product europium-labeled anti-diacetyl sickle enol monoclonal antibody. The europium labeling reagent is available from, but not limited to, Yoyour Biotech, Inc. in Shanghai.

Obtaining a sample reaction bottle containing europium-labeled anti-diacetyl grass fusarium enol monoclonal antibody freeze-dried powder: putting 0.25 mu g of europium-labeled anti-diacetyl sickle enol monoclonal antibody into a 3mL bayonet bottle, and pumping out by adopting a conventional freeze vacuum drying method to obtain europium-labeled anti-diacetyl sickle enol monoclonal antibody freeze-dried powder, and storing at 4 ℃ for later use.

The application of the flow lag immune time-resolved fluorescence rapid detection kit in detecting the sickle knife enol of a corn sample comprises the following steps:

establishing a relation curve of the ratio (T/C) of fluorescence intensity of a detection line and fluorescence intensity of a quality control line of a fluorescent test strip and the concentration of sickle knife enol of ribes diacetylenii:

(1) pretreatment is carried out on a corn sample which is negative to the detection of the diacetyl sickle knife fungus enol by High Performance Liquid Chromatography (HPLC), and the diacetyl sickle fungus enol is added to standard solutions with the concentrations of 0.5 mu g/mL, 0.25 mu g/mL, 0.125 mu g/mL, 0.0625 mu g/mL, 0.0312 mu g/mL and 0.0156 mu g/mL.

(2) Taking 100 mu L of each diacetyl sickle knife enol standard solution with each concentration, respectively adding the solutions into a sample reaction bottle, uniformly mixing, inserting fluorescent test strips, reacting for 6 minutes at 37 ℃, blotting residual liquid of a sample pad by using absorbent paper, immediately detecting by using a time-resolved fluorescence immunoassay analyzer (excitation wavelength: 365nm, detection wavelength: 615nm) to obtain fluorescence intensity values at a detection line (T) and a quality control line (C) on each fluorescent test strip, and thus obtaining the ratio (T/C) of the fluorescence intensity of each fluorescent test strip detection line to the fluorescence intensity of the quality control line;

(3) and establishing a standard curve by taking the concentration of the standard substance as an abscissa and the T/C value as an ordinate. The limit of quantitative detection is 0.10 ng/mL.

(4) The method comprises the steps of adding a standard substance of the diacetyl ribes sickle enol into a blank corn sample, wherein the concentration of the standard substance of the diacetyl ribes sickle enol is 0.1 mu g/mL, 0.05 mu g/mL and 0.01 mu g/mL, measuring the concentration of the diacetyl ribes sickle enol as a horizontal coordinate by using a fluorescent test strip, measuring the concentration of the diacetyl ribes sickle enol as a vertical coordinate by using HPLC, and the adding recovery rate of the method is 87.7-115.3%.

Example 2: quick detection kit for flow lag immune time resolution fluorescence of ribes diacetylenium sickle enol and application thereof

Quick detect reagent box of diacetyl grass sickle cell enol flow lag immunity time-resolved fluorescence, it includes fluorescence test paper strip, contains the europium mark anti diacetyl grass sickle enol monoclonal antibody freeze-dried product's sample reaction bottle, sample diluent and sample diluent straw, fluorescence test paper strip include the cardboard, the one side from the top down of cardboard pastes water absorption pad, detection pad and sample pad in proper order, adjacent each pad is in junction overlap connection, overlap length is 1mm, wherein: the absorbent pad is 18mm long and 3mm wide; the detection pad is 28mm long and 3mm wide; the sample pad was 15mm long and 3mm wide. The detection pad takes the nitrocellulose membrane as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, the coating amount of the quality control line is 150ng/cm, the detection line is coated with a diacetyl fusarium enol complete antigen (DAS-OVA), the coating amount of the DAS-OVA is 650ng/cm of the detection line, the distance between the detection line and the upper edge of the nitrocellulose membrane is 20mm, and the distance between the quality control line and the detection line is 10 mm.

Obtaining the fluorescent test strip:

(1) preparation of absorbent pad

Cutting the absorbent paper into pieces with the length of 18mm and the width of 3mm to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

preparing 0.25mg/mL coating solution from a coating buffer solution to a position 20mm away from a nitrocellulose membrane, transversely coating the coating solution on the nitrocellulose membrane in a membrane scratching manner to obtain a detection line, wherein the coating amount of the sickle cell enol complete antigen (DAS-OVA) required by each centimeter of detection line is 650ng, and drying the test line for 30 minutes at 40 ℃;

coating of quality control line:

preparing a coating buffer solution for the rabbit anti-mouse polyclonal antibody into a coating solution with the concentration of 0.25mg/mL at a position 6mm away from a detection line, transversely coating the coating solution on a nitrocellulose membrane in a line spraying manner to obtain a quality control line, wherein the coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter of the quality control line is 150ng, and then drying for 30min at 40 ℃;

the length of the nitrocellulose membrane is 28mm, and the width of the nitrocellulose membrane is 3 mm.

(3) Preparation of sample pad:

cutting the glass fiber membrane into pieces with the length of 15mm and the width of 3mm, soaking in a sealing solution, taking out, drying at 37 ℃ for 6 hours to obtain a sample pad, and then placing in a dryer for storage at room temperature;

(4) assembling the fluorescent test strip:

one side of the paperboard is sequentially stuck with a water absorption pad, a nitrocellulose membrane and a sample pad from top to bottom. And (3) overlapping and connecting the adjacent parts at the joint, wherein the overlapping length is 2mm, and thus the fluorescent test strip is obtained.

Obtaining the europium-labeled anti-diacetyl grass fusarenol monoclonal antibody:

1mg of the anti-diacetyl sickle knife enol monoclonal antibody is taken, washed repeatedly for 6 times by using 100mmol/L carbonate buffer solution with pH9.3, and then fully and uniformly mixed with 2mg of europium labeling reagent and stays overnight at 4 ℃. Then adding the europium-labeled anti-diacetyl sickle enol monoclonal antibody into a Sephadex G-50 chromatographic column of 1.9cm multiplied by 60cm, eluting with 50mmol/L Tris-HCl eluent containing 0.9% NaCl, collecting effluent liquid (1 ml/tube), measuring an absorbance value (A280nm) tube by tube, and combining peak tubes to obtain the target product europium-labeled anti-diacetyl sickle enol monoclonal antibody. The europium labeling reagent is available from, but not limited to, Yoyour Biotech, Inc. in Shanghai.

Obtaining a sample reaction bottle containing europium-labeled anti-diacetyl grass fusarium enol monoclonal antibody freeze-dried powder: putting 0.25 mu g of europium-labeled anti-diacetyl grass fusarium enol monoclonal antibody into a 3mL bayonet bottle, and pumping out by adopting a conventional freeze vacuum drying method to obtain europium-labeled anti-diacetyl grass fusarium enol monoclonal antibody freeze-dried powder, and storing the powder at 4 ℃ for later use.

The sample diluent is as follows: 0.30% volume fraction tween 20 in water.

a series of concentrations of a sickle knife fungus enol standard (1000, 200, 40, 8, 1.6, 0.32, 0.06ng/mL) was added to a blank corn sample. Pre-treating to obtain sample extractive solution, adding 200 μ L of the sample extractive solution into sample reaction bottle, mixing, inserting fluorescent test strip, reacting at 37 deg.C for 6 min, blotting residual liquid in sample pad with absorbent paper, immediately detecting with time-resolved fluoroimmunoassay analyzer (excitation wavelength: 365nm, measurement wavelength: 615nm), and measuring the ratio of fluorescence intensity of detection line to fluorescence intensity of quality control line (T)_x/C), establishment of T with orgin 8.5_xCurve of the relationship between the/C ratio and the corresponding toxin concentration.

Taking five corn samples to be detected, detecting the corn samples after pretreatment in the same way, and detecting the corn samples by using a time-resolved fluoroimmunoassay analyzer to obtain the ratio (T) of the fluorescence intensity of each fluorescent test strip detection line to the fluorescence intensity of the quality control line_x/C), and then substituting the fluorescence into a fluorescent test strip detection lineRatio of fluorescence intensity to quality-controlled fluorescence intensity (T)_xThe relationship curve of the concentration of the ketoenol of the sickle of ribes diacetylenium is obtained: the detection recovery rate is between 87.7% and 115.3%, and the correlation coefficient of the kit and the HPLC detection result reaches 0.988 (R2).

< 110 > institute of oil crops of Chinese academy of agricultural sciences

Rapid detection kit for flow lag immune time resolution fluorescence of < 120 > diacetyl ribes sickle enol

＜160＞ 4

＜210＞ 1

＜211＞ 351bp

＜212＞ DNA

< 213 > mice

＜400＞ 1

gaagtgcaac tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60

tcctgttcag cctccggatt cactttcaat tactatggca tgtcttgggt tcgccagact 120

ccagacaacc tcctggagtg ggtcgcaggc attagtagtg gtggttctta cacctattat 180

tctgacagtg tgaagggacg attcaccatc tccagagaca gtgccacgaa caccctgtac 240

ctgcaaatga ccagtctgaa gtctcaagac acagccatgt attattgtat tagactcccg 300

tttgggtcta tggactattg gggtcaagga accgcagtca ccgtctcctc a351

＜210＞ 1

＜211＞ 324bp

＜212＞ DNA

< 213 > mice

＜400＞ 2

caggctgttg tgactcagga acctgcactc accacatcac ctggtgaaac agtcacactc 60

acttgtcgct caagtactgg ggctgtaaca actggtaatt atgtcaactg ggtccaagag 120

aaaccagatc atttattcag tggtctaata ggtaatacca ataaccgagc tccaggtgtt 180

cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacagggaca 240

cagactgagg atgaggcaat atatttctgt gctctatggt acaccgacca tttggtgttc 300

ggtggaggaa ccaaattgac tgtc 324

＜210＞ 1

＜211＞ 117

＜212＞ PRT

< 213 > mice

＜400＞ 3

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Asn Leu Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Thr Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Ser Gln Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ile Arg Leu Pro Phe Gly Ser Met Asp Tyr Trp Gly Gln Gly Thr Ala

100 105 110

Val Thr Val Ser Ser

115

＜210＞ 1

＜211＞ 108

＜212＞ PRT

< 213 > mice

＜400＞ 4

Gln Ala Val Val Thr Gln Glu Pro Ala Thr Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Gly

20 25 30

Asn Tyr Val Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Ser Gly

35 40 45

Leu Ile Gly Asn Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Thr

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Thr Asp

85 90 95

His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105

Claims

1. The quick detection kit for the flow lag immune time resolution fluorescence of the sickle knife enol of ribes diacetylenii is characterized in that: it includes fluorescence test paper strip and contains the sample reaction bottle of the anti diacetyl grass fusarium enol monoclonal antibody freeze-dried product of europium mark, wherein: the fluorescent test strip comprises a lining plate, wherein a water absorption pad, a detection pad and a sample pad are sequentially adhered to one surface of the lining plate from top to bottom, the adjacent pads are overlapped and connected at the joint, the detection pad takes a nitrocellulose membrane as a base pad, a quality control line and a detection line are transversely arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the detection line is coated with a diacetyl ribes fusarenol complete antigen DAS-OVA; the anti-diacetylebergens fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO. C201881.

2. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the europium-labeled anti-diacetyl sickle enol monoclonal antibody is prepared according to the following method: dialyzing the anti-diacetyl sickle knife enol monoclonal antibody with a carbonate buffer solution, fully and uniformly mixing the dialyzed anti-diacetyl sickle knife enol monoclonal antibody with an europium-labeled reagent according to the mass ratio of 0.5-2: 1, standing overnight, separating the europium-labeled anti-diacetyl sickle knife enol monoclonal antibody through a Sephadex G-50 chromatographic column, eluting, and collecting a target product.

3. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the length of the water absorption pad in the fluorescent test strip is 15-20 mm, and the width of the water absorption pad is 3-4 mm; the detection pad is 25-30 mm long and 3-4 mm wide; the sample pad is 12-18 mm long and 3-4 mm wide, and the overlapping length of each adjacent pad is 1-3 mm.

4. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the distance between a detection line on a detection pad in the fluorescent test strip and the upper edge of the nitrocellulose membrane is 15-20 mm, and the distance between a quality control line and the detection line is 5-10 mm; the sample reaction bottle is a 1-5 mL bayonet bottle.

5. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: 480-1000 ng of diacetyl ribes sickle knife enol complete antigen coating amount required by each centimeter detection line on the detection pad in the fluorescent test strip; coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter of quality control line is 100-900 ng; the content of the europium-labeled anti-diacetylebergen fusarium enol monoclonal antibody freeze-dried product in the sample reaction bottle is 0.2-1.0 mu g.

6. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the fluorescent test strip is obtained by adopting the following method:

(1) cutting the absorbent paper into absorbent pads with required sizes;

(2) preparation of a detection pad:

preparing a DAS-OVA antigen coating solution with the concentration of 0.125-0.8 mg/mL by using a diacetyl sickle enol complete antigen DAS-OVA, transversely coating the DAS-OVA antigen coating solution on a nitrocellulose membrane in a membrane scratching mode to obtain a detection line, and drying the detection line for 30-60 minutes at 37-40 ℃; preparing a coating solution with the concentration of 0.1-1.0 mg/mL by using a rabbit anti-mouse polyclonal antibody, transversely coating the coating solution on a nitrocellulose membrane in a membrane scratching mode to obtain a quality control line, and drying the nitrocellulose membrane for 30-60 minutes at 37-40 ℃;

(3) preparation of sample pad:

putting the glass fiber membrane into the sealing liquid for completely soaking, taking out the glass fiber membrane, drying the glass fiber membrane for 3-6 hours at 37-40 ℃ to obtain a required sample pad, and storing the sample pad in a dryer at room temperature;

(4) assembling the fluorescent test strip:

7. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the coating buffer solution used in the preparation of the sickle knife enol complete antigen DAS-OVA coating solution in the preparation of the fluorescent test strip is as follows: every 10mL contains bovine serum albumin 0.1g, sodium chloride 0.08g, potassium chloride 0.002g, sodium azide 0.002g, disodium hydrogen phosphate dodecahydrate 0.029g, and potassium dihydrogen phosphate 0.002 g;

8. The reagent kit for rapid detection of flow delay of sickle knife enol of ribes diacetylenium according to claim 1, which is characterized in that: the quick detection kit for flow lag immunity time-resolved fluorescence of ribes diacetylenium sickle enol further comprises a sample diluent and a sample diluent suction tube, wherein the sample diluent is a Tween 20 aqueous solution with the volume fraction of 0.01-0.30%.

9. The use of the fast detection kit for flow delay of sickle enol of ribes diacetylenium according to claim 1 for the detection of the content of sickle enol of ribes diacetylenium: adding a sample solution to be detected into a sample reaction bottle, uniformly mixing, inserting a fluorescent test strip, reacting at 37 ℃ for 6-10 minutes, and detecting by using a time-resolved fluorescence tester to obtain the ratio of the fluorescence intensity value of a detection line (T) on the fluorescent test strip to the fluorescence intensity value of a quality control line (C); based on a relation curve between the ratio (T/C) of fluorescence intensity of a fluorescence test strip detection line to fluorescence intensity of a quality control line and concentration of the diacetyl ribes sickle knife enol, the content of the diacetyl ribes sickle knife enol in the sample liquid to be detected is obtained.

10. Use according to claim 9, characterized in that: the relation curve of the ratio (T/C) of the fluorescence intensity of the fluorescence test strip detection line to the fluorescence intensity of the quality control line to the concentration of the sickle knife enol of the ribes is obtained by adopting the following method: