CN111500546A

CN111500546A - Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card

Info

Publication number: CN111500546A
Application number: CN202010378256.1A
Authority: CN
Inventors: 边超; 姚譞; 樊敏伟
Original assignee: Shanghai Yimiao Hezhen Biomedical Technology Co ltd; Shanghai Tcm Technology Co ltd
Current assignee: Shanghai Yimiao Hezhen Biomedical Technology Co ltd; Shanghai Tcm Technology Co ltd
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-07

Abstract

The invention provides a cell strain secreting four subtype antibodies against aflatoxin, an antibody secreted by the cell strain and an immunochromatography detection card. The cell strain is classified and named as an anti-aflatoxin hybridoma cell strain 8B11, and the preservation number is CCTCC NO: c2019314, the preservation date is 2019, 12 months and 4 days, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university, Wuhan City, China. The antibody secreted by the cell strain 8B11 secreting the anti-aflatoxin four-subtype antibody can be combined with high affinity and can simultaneously recognize the four main aflatoxin subtypes specified in Chinese pharmacopoeia, namely B1, B2, G1 and G2, thereby providing a foundation for developing a total aflatoxin rapid detection product. In addition, the invention provides a convenient and rapid pretreatment method of the traditional Chinese medicinal materials, is matched with an immunochromatography detection card based on antigen-antibody reaction, is used for rapidly detecting aflatoxin of the traditional Chinese medicinal materials, has the advantages of rapidness, convenience and low cost, and can be popularized in a large scale.

Description

Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card

Technical Field

The invention relates to the field of hybridoma cell strains for resisting four subtype antibodies of aflatoxin, in particular to a cell strain secreting four subtype antibodies of aflatoxin, secreted antibodies of the cell strain and an immunochromatography detection card.

Background

Aflatoxins (AFT) are a class of chemical structure-like compounds, all of which are derivatives of dihydrofurocoumarin, and about 20 are named as B1, B2, G1, G2, M1, M2, GM, P1, Q1, toxol, etc. Aflatoxins are secondary metabolites produced primarily by aspergillus flavus and aspergillus parasiticus, and the probability of aflatoxins occurring in foods and feeds in hot and humid areas is highest. They are present in soil, animals and plants, various nuts, and are particularly easy to pollute grain and oil products such as peanuts, corns, rice, soybeans, wheat and the like. In a carcinogen list published by international cancer research institution of world health organization in 2017, the aflatoxin site is a category of carcinogens, and is a highly toxic substance, wherein B1 has the highest toxicity and the carcinogenicity is the highest. After animals eat the aflatoxin-contaminated feed, trace amounts of toxins can be detected in the liver, kidneys, muscles, blood, milk and eggs. Aflatoxin B1(Aflatoxin B1 abbreviated as AFB1) is a derivative of dihydrofuran phthalazone containing one difuranic ring and one phthalazone (coumarin). Aflatoxin B1 is the most common and most harmful in natural foods, and the national quality control Bureau stipulates that aflatoxin B1 is one of the necessary inspection items of most foods. In the field of traditional Chinese medicines, 19 traditional Chinese medicinal materials are specified in 2015 edition of Chinese pharmacopoeia for aflatoxin inspection, 14 varieties are added compared with 2010 edition of Chinese pharmacopoeia, and the method specifically comprises the following steps: polygala root, Chinese date, nutmeg, cassia seed, malt, dried orange peel, rangooncreeper fruit, platycladi seed, boat-fruited sterculia seed, lotus seed, peach seed, betel nut, spina date seed, coix seed, leech, earthworm, scorpion, centipede and stiff silkworm. The aflatoxin B1 is required to be not more than 5 mug/kg; the total amount of aflatoxin B1, aflatoxin G2, aflatoxin G1 and aflatoxin B2 should not exceed 10 μ G/kg.

The fluorescence immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) as a stationary phase, a test solution as a mobile phase, a fluorescence labeled antibody or antigen fixed on a connecting pad, and an analyte to be analyzed moves on the chromatography strip through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with a plurality of antigenic determinants, a sandwich type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is combined with a coating antibody to form a sandwich type double-antibody sandwich when reaching a detection line. For small molecule antigens (veterinary drugs, prohibited drugs and the like) with only a single epitope, after the small molecule antigens to be detected are combined with the fluorescence labeling antibody, the small molecule antigens are difficult to be combined with the coating antibody on the detection line due to steric hindrance. Therefore, the small molecule analyte with single epitope is mostly detected by using the competitive immunochromatography.

The fluorescence immunochromatographic assay technology is very suitable for detecting aflatoxin, has the advantages of high sensitivity, good stability, low cost, high speed, low interference of natural fluorescence and the like, becomes a hotspot of food quality safety rapid detection and analysis research, and is a detection technology which is easy to use on site instantly. Currently, labels used for fluorescence immunoassay mainly include fluorescein, quantum dots, upconversion nanoparticles, and the like.

The core of a detection product developed based on a fluorescence immunochromatographic method is a monoclonal antibody for specifically recognizing aflatoxin, and the combination sensitivity and specificity of the antibody determine the quality of the detection kit, so that the development of a good aflatoxin monoclonal antibody is a key premise for the development of the detection kit. The existing rapid detection products for detecting aflatoxin widely existing in the market are all colloidal gold or fluorescence immunochromatographic detection cards for detecting aflatoxin B1. The monoclonal antibody reagent used in the method specifically recognizes aflatoxin B1 subtype, and has no recognition on other subtypes. However, if a rapid detection product for detecting total aflatoxins, namely four aflatoxin subtypes of B1, B2, G1 and G2, is to be developed, a monoclonal antibody reagent capable of simultaneously recognizing the four aflatoxins needs to be obtained first, and the traditional Chinese medicine is pretreated, so that the aflatoxins in the traditional Chinese medicine are rapidly and fully dissolved in a neutral liquid medium suitable for being combined with the antibody reagent.

The prior aflatoxin pretreatment method specified in pharmacopoeia comprises the following steps: taking about 15g of sample powder (passing through a No. two sieve), precisely weighing, adding 3g of sodium chloride, placing in a homogenizing bottle, precisely adding 75ml of 70% methanol solution, stirring at a high speed for 2 minutes (the stirring speed is more than 11000 r/min), centrifuging for 5 minutes (the centrifugation speed is 2500 r/min), precisely measuring 15ml of supernatant, placing in a 50ml measuring flask, diluting with water to scale, shaking uniformly, filtering with a microporous filter membrane (0.45 mu m), measuring 20.0ml of subsequent filtrate, passing through an immunoaffinity column at a flow rate of 3ml per minute, eluting with 20ml of water, discarding the eluent, allowing air to enter the column, extruding water out of the column, eluting with an appropriate amount of methanol, collecting the eluent, placing in a 2ml measuring flask, diluting with methanol to scale, and shaking uniformly to obtain the reagent. (2015 edition "Chinese pharmacopoeia)". The method is developed by matching with subsequent liquid chromatography detection, and is not suitable for matching with an immunochromatography rapid detection method. The main problems are that: 1. finally, detecting that the content of methanol in the sample is too high, so that the antigen-antibody binding reaction is influenced; 2. the immunoaffinity column is adopted for purification, so that although a large amount of impurities which can influence chromatographic detection are removed, the detection cost is improved, and the treatment steps and the treatment time are increased; 3. the treatment process is long in time consumption and is relatively complicated.

In summary, the traditional Chinese medicine aflatoxin is rapidly detected, and a monoclonal antibody capable of simultaneously recognizing four aflatoxins and a traditional Chinese medicine pretreatment method suitable for matching with an immunochromatography rapid detection method are needed.

Disclosure of Invention

The invention provides a cell strain secreting four subtype antibodies against aflatoxin, an antibody secreted by the cell strain and an immunochromatography detection card in order to overcome the defects of the prior art. Monoclonal antibodies resisting aflatoxins B1, B2, G1 and G2 are generated by constructing hybridoma cell strains and purifying the monoclonal antibodies, and based on the monoclonal antibodies, an immunochromatography detection card is prepared to realize rapid detection of aflatoxins B1, B2, G1 and G2 of Chinese medicinal materials.

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

the first aspect of the invention provides a cell strain secreting four subtype antibodies against aflatoxin, which is classified and named as an anti-aflatoxin hybridoma cell strain 8B11 with the preservation number of CCTCC NO: c2019314, the preservation date is 2019, 12 months and 4 days, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university, Wuhan City, China.

The second aspect of the invention provides an antibody secreted by the cell strain secreting four subtypes of anti-aflatoxin antibodies, and the preparation method of the antibody comprises the following steps:

step one, animal immunization: mixing aflatoxin B1 hapten with an immunologic adjuvant, emulsifying, and immunizing the experimental animal by subcutaneous injection; after three times of immunization, aflatoxin B1 hapten is dissolved in PBS buffer solution, and then intraperitoneal injection is carried out to carry out memory stimulation;

step two, construction of hybridoma cell strains: myeloma cells and splenocytes are mixed according to a proportion, then preheated fusion agent is added at room temperature for fusion, and the fused cells are placed in an incubator for culture after being processed to obtain cell strains secreting four subtype antibodies against aflatoxin;

step three, performing monoclonality on the hybridoma cell strain by using a limiting dilution method;

step four, producing the antibody: inoculating hybridoma cells to the experimental animal injected with pristane, and extracting ascites containing the antibody from the abdominal cavity after inoculating for 8-12 days;

and step five, purifying the antibody.

Further, the injection dose of the aflatoxin B1 hapten in the first step is 0.1 mg/time/mouse.

Further, in step two, the myeloma cells are in logarithmic growth phase.

Further, in step two, the ratio of myeloma cells to splenocytes is 1:10 or 1: 5.

Further, the fusion agent in the second step is a 45% polyethylene glycol (PEG) solution containing 5% DMSO, and the fusion time is 60-120 s.

Further, the specific steps of antibody purification are:

(1) diluting the extracted ascites with cold PBS solution, centrifuging, and removing precipitate;

(2) slowly dropwise adding saturated ammonium sulfate solution into the supernatant at 4 ℃, and stirring while adding until the concentration of ammonium sulfate in the supernatant is 50%;

(3) placing the treated supernatant in ice for 30-60 min, centrifuging, removing the supernatant, dissolving the precipitate in Tris-HCl buffer solution, and dialyzing;

(4) after dialysis, the precipitate was centrifuged, and then the supernatant was diluted to separate and purify the antibody by column chromatography.

The third aspect of the invention provides an immunochromatography detection card for rapidly detecting total aflatoxins of traditional Chinese medicines, and the specific preparation method comprises the following steps:

activating fluorescent microspheres by using an activating agent, then adding the antibody or rabbit IgG, and then oscillating, centrifuging and sealing to obtain an antibody coupled fluorescent microsphere or rabbit IgG coupled fluorescent microsphere solution;

step two, dropwise adding a pretreatment solution on the sample pad, and dropwise adding a conjugate diluent on the bonding pad;

step three, uniformly mixing the antibody coupling fluorescent microsphere solution prepared in the step one and the rabbit IgG coupling fluorescent microsphere solution, and spraying the mixture onto a bonding pad;

step four, respectively diluting aflatoxin B1 hapten and goat anti-rabbit IgG with PBS, respectively adding 1% of antibody additive, scribing on an NC membrane, and scribing 1 mul/cm on each T, C line;

and step five, drying the combination pad and the NC membrane, then sequentially pasting the NC membrane, the combination pad, the sample pad and the absorbent paper on a bottom plate, and cutting into test strips with certain widths, namely the immunochromatography detection card.

Further, the activating agent in the first step comprises the following concentration solutions: 50mg/ml Sulfo-NHS solution and 50mg/ml EDC solution or 50mg/ml EDC.

Further, the preparation method of the conjugate diluent in the second step comprises the following steps: after 0.7g Tx-100, 180ml water, 0.242g Tris, 10g sucrose and 2g BSA were completely dissolved, the pH was adjusted to 7.4, water was added to 200ml, filtered and stored at 4 ℃.

Further, the preparation method of the pretreatment solution in the second step comprises the following steps: after 0.6057g Tri, 0.5g BSA, 1g Tween 20, 0.073g EDTA and 20. mu.l Proclin 300 were completely dissolved, the pH was adjusted to 7.4, water was added to 100ml, and the mixture was stored at 4 ℃.

Further, before detecting aflatoxin in the traditional Chinese medicinal materials by using an immunochromatography detection card, the traditional Chinese medicinal materials need to be pretreated, and the specific treatment steps are as follows:

(1) accurately weighing 15g of medicinal material powder to be measured, adding 40-60ml of 70% methanol solution, and stirring at high speed for 1-2 minutes by using a homogenizer;

(2) then centrifuging the solution stirred in the step (1) at room temperature or filtering by using quantitative filter paper;

(3) and collecting the supernatant or the filtrate, adding a phosphate buffer solution, and uniformly mixing to obtain the processed sample to be detected.

Further, the volume ratio of the supernatant or filtrate to the phosphate buffer is 100: 200-800.

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

the antibody secreted by the cell strain 8B11 secreting the anti-aflatoxin four-subtype antibody can be combined with high affinity and can simultaneously recognize the four main aflatoxin subtypes specified in Chinese pharmacopoeia, namely B1, B2, G1 and G2, thereby providing a foundation for developing a total aflatoxin rapid detection product. In addition, the invention provides a convenient and rapid pretreatment method of the traditional Chinese medicinal materials, is matched with an immunochromatography detection card based on antigen-antibody reaction, is used for rapidly detecting aflatoxin of the traditional Chinese medicinal materials, has the advantages of rapidness, convenience and low cost, and can be popularized in a large scale.

Drawings

The invention discloses a cell strain secreting four subtype antibodies against aflatoxin, which is preserved and classified and named as an anti-aflatoxin hybridoma cell strain 8B11, and the preservation number is CCTCC NO: c2019314, the preservation date is 2019, 12 months and 4 days, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university, Wuhan City, China.

FIG. 1 is a graph of the quantitation of the total aflatoxin antigen by monoclonal antibody 6B12 in one embodiment of the present invention;

FIG. 2 is a bar graph showing the recognition reactivity of monoclonal antibody 6B12 to four different aflatoxin subtypes (B1, B2, G1, G2) in one embodiment of the present invention;

FIG. 3 is a quantitative standard curve of the immunochromatographic assay card for detecting total aflatoxins in one embodiment of the present invention.

Detailed Description

The invention relates to a cell strain secreting four subtype antibodies against aflatoxin, an antibody secreted by the cell strain and an immunochromatography detection card.

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Unless otherwise specified, the reagents and materials used in the experimental procedures of the present invention are conventional commercial reagents and materials, the standard experimental methods are used (for details, refer to "antibody technology experimental guidance" (e. harlo science publishers 2005) and "antibody preparation and use experimental guidance" (g.c. howland science publishers 2010), and the mentioned academic terminology and its english abbreviations are used according to the rules of "immunological nouns" of the national science and technology noun committee (science publishers 2008) ", unless otherwise specified. The following examples relate to reagents and apparatus as follows:

aflatoxin B1 hapten (aflatoxin B1 BSA conjugate, abbreviated as AFB1-BSA) was purchased from beijing fubo biotechnology ltd;

aflatoxins B1, B2, G1, G2 controls were purchased from beijing solibao technologies ltd;

aflatoxin mixed reference substances were purchased from the institute of food and drug testing, china;

the mouse myeloma cell line SP2/0 was purchased from cell bank of Chinese academy of sciences;

the fluorescent microspheres were purchased from Nanjing micro-assay Biotechnology Ltd;

the fluorescence immunoassay analyzer was purchased from Hangzhou Gelunkun technologies, Inc.

Example one

The embodiment provides a cell strain secreting four subtype antibodies against aflatoxin, which is preserved and named as an anti-aflatoxin hybridoma cell strain 8B11, and the preservation number is CCTCC NO: c2019314, the preservation date is 2019, 12 months and 4 days, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university, Wuhan City, China.

The construction method of the cell strain secreting four subtype antibodies against aflatoxin comprises the following steps:

step one, animal immunization:

experimental animals female 6-week-old mice of Balb/c strain were used. An immunization process: AFB1-BSA was mixed with Freund's complete adjuvant, emulsified and injected subcutaneously at multiple sites for immunization. The dose of AFB1-BSA was 0.1 mg/dose/mouse. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the second immunization. The interval time of each immunization was 3 weeks, and 3 immunizations were performed. After the third immunization, the mice were reminiscent stimulated before cell fusion, and 0.1mg of antigen was dissolved in 0.5ml of PBS buffer and injected intraperitoneally. Cell fusion and hybridoma construction were performed 3 days after recall stimulation.

Step two, hybridoma cell strain construction:

preparing a feeder cell suspension one day before cell fusion, wherein 5-8 × 10 can be obtained from one mouse⁶Peritoneal macrophages, when mouse thymocytes were used as feeder cells, cell concentration was 5 × 10⁶Perml, mouse splenocytes 1 × 10⁶Mouse fibroblast (3T3)1 × 10/ml⁵Each 100. mu.l/well of the suspension was used.

(1) Taking logarithmic growth myeloma cells SP2/0, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, suspending the cells with incomplete culture solution and counting, taking the required number of cells, washing 2 times with incomplete culture solution (RPMI 1640).

(2) At the same time, immune splenocyte suspension was prepared and washed 2 times with incomplete medium.

(3) Myeloma cells and splenocytes were mixed together at a ratio of 1:10 or 1:5 and washed 1 time with incomplete culture medium in a 50ml plastic centrifuge tube at 1200rpm for 8 minutes.

(4) Discard the supernatant, and use the dropper to suck up the residual liquid, so as to avoid affecting the concentration of PEG.

(5) Lightly flick the bottom of the tube to loosen the cell pellet.

(6) Fusion at room temperature: preheated 1ml 45% PEG (Merek, MW 4000) containing 5% DMSO was added within 30 seconds while stirring, and after 90 seconds of action, preheated incomplete culture broth was added to terminate the PEG action, and 1ml, 2ml, 3ml, 4ml, 5ml and 10ml were added every 2 minutes.

(7) Centrifuge, 800rpm, 6 minutes.

(8) The supernatant was discarded and first suspended gently with about 6ml of 20% calf serum RPMI1640, and the cells were not blown hard to scatter.

(9) Complete medium was added to 10ml of a 96-well plate depending on the number of 96-well plates used.

(10) The fused cell suspension was added to a 96-well plate containing feeder cells at 100. mu.l/well, 37 ℃ with 5% CO₂And (5) incubator culture.

(11) After 24 hours of fusion, HAT selection medium (1 ml is added into 50ml of 20% calf serum complete medium), then at 37 deg.C,5％CO₂Culturing for 3-7 days under the condition to obtain the cell strain secreting four subtype antibodies against aflatoxin.

Example two

This example provides antibodies against four aflatoxin subtypes (B1, B2, G1, G2) prepared using the cell lines constructed in example one, and the preparation process is as follows:

step one, monoclonal cloning of hybridoma cell lines (limiting dilution method):

(1) preparing a feeder cell suspension, wherein 5-8 × 10 can be obtained from one mouse⁶Peritoneal macrophages, when mouse thymocytes were used as feeder cells, cell concentration was 5 × 10⁶Perml, mouse splenocytes 1 × 10⁶Mouse fibroblast (3T3)1 × 10/ml⁵Each 100. mu.l/well of the suspension was used.

(2) Counting positive hole cells and regulating the cell number to 1-5 × 10³/ml。

(3) 130 cells were taken and placed in 6.5ml complete medium containing feeder cells, i.e. 20 cells/ml, 100. mu.l/well plus A, B, C triple rows of 2 cells per well. The remaining 2.9ml of cell suspension was supplemented with 2.9ml of complete medium containing feeder cells, the number of cells was 10 per ml, and D, E, F rows were added at 100. mu.l/well, for 1 cell per well. The remaining 2.2ml of cell suspension was supplemented with 2.2ml of complete medium containing feeder cells, 5 cells/ml, 100. mu.l/well, and G, H two rows of 0.5 cells per well.

(4) After 4-5 days of culture, small cell clones were visible on an inverted microscope and 200. mu.l/well of complete culture medium was added.

(5) And at 8-9 days, cell cloning can be seen by naked eyes, and antibody detection is carried out in time. Note: primary cloned hybridoma cells require the addition of HT to the complete medium.

(6) Positive hybridoma cells were cloned 3 times in succession using limiting dilution. And (3) constructing a stable cell strain, and performing expanded culture and cryopreservation.

Step two, antibody production:

(1) and (3) injecting 0.5ml of pristane into the abdominal cavity of the mouse, and planting cells within 1-9 weeks after injection.

(2) Collecting hybridoma cells in logarithmic growth phase, washing with incomplete culture solution once, and centrifuging at 1000r/min for 10 min.

(3) Sampling, staining with trypan blue, counting viable cells, and preparing again with incomplete culture medium to obtain 1.0 × 10⁷Cells/ml suspension.

(4) Mice injected with pristane were inoculated with hybridoma cells, and 1ml (containing 1.0 × 10) per abdominal cavity was injected⁷Individual cells/ml).

(5) The tumor volume is maximum about 10 days after inoculation, and ascites can be extracted from the abdominal cavity at the moment and taken 1 time every 1-3 days. Serum can be isolated from the axillary artery or from the heart after collection.

Step three, purifying the monoclonal antibody

(1) The ascites of the mice were diluted 4-fold with cold PBS and then treated at 1 × 10⁵Centrifuging for 30min, and removing precipitate.

(2) Saturated ammonium sulfate solution was slowly added dropwise to the supernatant at 4 ℃ while stirring, so that the final solution had a concentration of 50% ammonium sulfate.

(3) The solution is placed in ice for 30 min-60 min, then centrifuged for 10min at 5000r/min, and the supernatant is removed.

(4) The pellet was dissolved in Tris-HCl buffer (40 mMol/L NaCl) (solution may be cloudy).

(5) The mixture was put into a dialysis bag and dialyzed against Tris-HCl buffer (20 mMol/L NaCl) to remove the salts.

(6) And centrifuging to remove the precipitate.

(7) After dilution of the solution (1:100 or more fold dilution), the protein content was measured at 280nm and estimated: 1a280unit ═ 0.8mg protein. Generally, about 25mg to 36mg of total protein per ml of ascites fluid is contained.

(8) Passing through a DEAE-cellulose column, wherein the height of the cellulose column is 40cm, balancing with 20 mMol/L NaCl Tris buffer solution, diluting a dialysis sample with the same amount of the Tris buffer solution, feeding the sample into a column bed at the speed of 1 ml-2 ml/min, eluting with a linear NaCl gradient, eluting most of monoclonal IgG in 40 mMol/L and 80 mMol/L NaCl, eluting with few monoclonal antibodies in 120 mMol/L-150 mMol/L NaCl, measuring OD280nm, collecting protein peaks, and storing the monoclonal IgG (6B12) for later use.

EXAMPLE III

In this embodiment, a competitive E L ISA method is used to detect the quantitative recognition of monoclonal IgG to total aflatoxin, and the specific steps are as follows:

(1) coating antigen, namely preparing the antigen (B1-BSA) into coating solution with a certain concentration (3 mu g/ml) by using carbonate buffer solution with pH9.6, mixing the coating solution evenly, adding a 96-hole E L ISA detection plate, placing the solution in a refrigerator at 4 ℃ for one night at 100 mu l/hole.

(2) And (3) sealing: plates were washed, added with blocking solution (0.5% BSA in PBS), 200. mu.l/well, and incubated for 2 hours in a 37 ℃ incubator.

(3) Primary antibody (test sample): and (5) washing the plate. Monoclonal antibody 6B12 was diluted to a concentration of 2 μ g/ml with PBS, and mixed with serially diluted B1 standards at a ratio of 1:1 mix, shake slightly, 50. mu.l/well, add immediately to the microplate, incubate for 1h in a 37 ℃ incubator.

(4) Adding a secondary antibody: and (5) washing the plate. Diluted secondary antibodies (commercial goat anti-mouse Ig-HRP, e.g.: ab6789, abcam, 1:2000 dilution) were added to the wells at 100. mu.l/well. Incubate at 37 ℃ for 1 hour.

(5) Color development: and (5) washing the plate. The TMB substrate was dispensed and added to the wells at 100. mu.l/well. After developing for 5-10 min, adding 100 mul/hole of 2M sulfuric acid to terminate the reaction.

(6) And (3) detection: and detecting the absorbance OD value of the solution at 450nm by using a microplate reader, and then analyzing the data.

As can be seen from FIG. 1, the aflatoxin B1-BSA coated at 3. mu.g/ml and the competitive antibody 6B12 at 2. mu.g/ml, the total aflatoxin concentration which can be quantitatively detected is in the range of 0.03-3 ng/ml.

Example four

In this example, the detection method of E L ISA is adopted to determine the identification of monoclonal antibody 6B12 to different subtypes of aflatoxin (B1, B2, G1 and G2), and comprises the following steps:

(1) coating antigen, namely preparing the antigen (B1-BSA) into coating solution with a certain concentration (4 mu g/ml) by using carbonate buffer solution with pH9.6, mixing the coating solution evenly, adding a 96-hole E L ISA detection plate, placing the solution in a refrigerator at 4 ℃ for one night at 100 mu l/hole.

(3) Primary antibody (test sample): and (5) washing the plate. The treated primary antibody solution, namely the purified monoclonal antibody 6B12 (eluent in the 8 th step in the third step of the example II) was mixed with aflatoxins of different subtypes and incubated at 37 ℃ for 1 hour, wherein the aflatoxin concentration is 0.05mg/ml and the antibody concentration is 0.1 mg/ml. After incubation, the solution was added to the wells at 100. mu.l/well. Incubate at 37 ℃ for 2 hours.

(4) Adding a secondary antibody: plates were washed and diluted secondary antibody (commercial goat anti-mouse Ig-HRP, e.g.: ab6789, abcam, diluted 1: 2000) was added to the wells at 100. mu.l/well. Incubate at 37 ℃ for 1 hour.

(5) Color development: the plate was washed, the TMB substrate was dispensed and added to the wells at 100. mu.l/well. After developing for 5-10 min, adding 100 mul/hole of 2M sulfuric acid to terminate the reaction.

As can be seen from FIG. 2, monoclonal antibody 6B12 has better recognition for aflatoxins of four subtypes, B1, B2, G1 and G2.

EXAMPLE five

The embodiment provides an immunochromatography detection card for rapidly detecting total aflatoxin of a traditional Chinese medicine, and a preparation method of the immunochromatography detection card comprises the following steps:

step one, preparing antibody coupled fluorescent microspheres:

1. activated microspheres

1.1. 275. mu.l of 0.1M NaH was added to each of 2 1.5ml centrifuge tubes₂PO₄Solution (pH6.2), 50. mu.l fluorescent microspheres, and mix well.

1.2. Formulating activators

50mg/ml Sulfo-NHS solution +50mg/ml EDC solution;

50mg/ml Sulfo-NHS solution +50mg/ml EDC. HCl solution.

1.3. Microsphere activation

Adding 6 mu l of Sulfo-NHS solution into an EP tube No. 1, uniformly mixing, immediately adding 22 mu l of EDC solution, and uniformly mixing; add 6. mu.l of sulfo-NHS solution into EP tube No. 2, mix well, add 22. mu.l of EDC. HCl solution immediately, mix well, shake 20min at 37 ℃, 250 r.

1.4. Centrifuging at 15000 × g for 10min, and removing supernatant.

EP tube No. 1.5.1 repeats steps 1.3 and 1.4, activating the microspheres a second time.

1.6. 250 μ l of 0.1M HEPES (pH 6.5) was added to each EP tube and mixed well.

1.7. Ultrasonic: the power is 100W, the work is 4s, the interval is 2s, and the work times are 50.

2. Coupling of

2.1.250 μ l of activated microspheres were added 40 μ g of 6B12 antibody (tube 1) and 40 μ g of rabbit IgG (tube 2), respectively.

Shaking at 2.2.37 deg.C for 1.5h, 250 r.

2.3. Centrifuging at 15000 × g for 10min, and removing supernatant.

3. Sealing of

3.1. Mu.l of 1% BSA solution (0.1g BSA made up to 10ml with 0.1M HEPES pH 6.5) was added, filtered and mixed well.

Shaking at 3.2.37 deg.C for 40min, 250 r.

3.3. Centrifuging at 15000 × g for 10min, and removing supernatant.

3.4. Adding 360 ul and 720 ul of conjugate storage solution respectively and mixing evenly.

3.5. Ultrasonic: the power is 100W, the work is 4s, the interval is 2s, the work frequency is 50 times, and the storage is carried out at 4 ℃.

Step two, preparing a fluorescence immunochromatographic test strip:

(1) the conjugate pad was cut to 11 × 300mm, sample pad 11 × 300mm, absorbent paper 16 × 300mm, NC film 25 × 300mm, and base plate 60 × 300mm gauge.

(2) Sample pad: the sample pad pretreatment solution, 70. mu.l/cm, was dropped on the sample pad, and dried at 37 ℃.

(3) Combining the pads: the conjugate diluted solution (30. mu.l/cm) was dropped onto the conjugate pad, and dried at 37 ℃.

(4) Mu.l of the fluorescently-labeled 6B12 antibody solution (111.1. mu.g/ml) and 30. mu.l of the rabbit IgG solution (55.56. mu.g/ml) were put into a centrifuge tube, mixed, and sprayed onto the pad at 2. mu.l/cm.

(5) B1-BSA and goat anti-rabbit IgG were diluted to 1.5mg/ml and 0.2mg/ml with PBS, respectively, and 1% antibody additive was added thereto and drawn on NC membranes, and lines T, C were drawn at 1. mu.l/cm each.

(6) The conjugate pad and NC membrane were dried at 37 ℃.

(7) And sequentially sticking the NC film, the combination pad, the sample pad and the absorbent paper on the bottom plate, and cutting into test strips with the width of 4mm, namely the immunochromatography detection card.

The preparation method of the conjugate diluent comprises the following steps: after 0.7g Tx-100, 180ml water, 0.242g Tris, 10g sucrose and 2g BSA were completely dissolved, the pH was adjusted to 7.4, water was added to 200ml, filtered and stored at 4 ℃.

The preparation method of the sample pad treatment solution comprises the following steps: after 0.6057g Tri, 0.5g BSA, 1g Tween 20, 0.073g EDTA and 20. mu.l Proclin 300 were completely dissolved, the pH was adjusted to 7.4, water was added to 100ml, and the mixture was stored at 4 ℃.

EXAMPLE six

The embodiment provides a traditional Chinese medicine pretreatment method suitable for aflatoxin immunochromatography rapid detection, which comprises the following steps:

(1) accurately weighing 15g of powder of a medicinal material to be detected in a beaker, adding 3g of sodium chloride (non-oily medicinal material may not be added) into an oily medicinal material, adding 40-60ml of 70% methanol solution, and stirring at a high speed for 1-2 minutes by using a homogenizer (the stirring speed is greater than 11000 r/min).

(2) Centrifuging for 5 minutes (the centrifugation speed is 2500 rpm) at room temperature (20-25 ℃), filtering with quantitative filter paper if no centrifugal equipment is used, and squeezing with a glass stirring rod if the medicinal material has too strong hygroscopicity.

(3) Collect the supernatant or filtrate and accurately pipette 100. mu.l into the centrifuge tube.

(4) Adding 200-800 μ l phosphate buffer solution (recovered to room temperature in advance) into the centrifuge tube, and mixing uniformly to obtain the sample to be detected. The diluted sample to be tested should be tested within 6 hours after preparation.

EXAMPLE seven

In this embodiment, the fluorescence immunochromatographic detection card provided in the fifth embodiment is used for detecting total aflatoxins in a traditional Chinese medicine sample, and the specific steps are as follows:

(1) taking out the detection card and laying on a table.

(2) And (3) sucking 100 mu l of a sample to be detected by using a pipette, vertically dripping the sample into the sample adding hole, starting timing after sample adding, and reacting for 15 minutes.

(3) Connecting the fluorescence immunoassay instrument with a power supply, shifting a power switch to a minus position, starting the fluorescence immunoassay instrument, inserting a detection card into the fluorescence immunoassay instrument (note: a sample adding hole is inward), pressing a detection key, displaying a quantitative detection result on a liquid crystal display screen of the instrument, and printing by pressing a printing key to obtain a paper detection report.

(4) Standard curve: before the instrument is used, a quantitative standard curve is drawn firstly, and the detection result is compared with the quantitative standard curve to calculate the final total aflatoxin content. Method for making standard curve: the total aflatoxin standard control was formulated in phosphate buffered saline as reference solutions of 9ng/ml, 3ng/ml, 1ng/ml, 0.33ng/ml, 0.11ng/ml and 0. The corresponding fluorescence value (T/C) is obtained by adopting the detection method of the detection card, and a standard curve is drawn (as shown in figure 3).

(5) Preparation of traditional Chinese medicinal material samples for experiments: the aflatoxin-negative traditional Chinese medicine sample (platycladi seed, Chinese date, nutmeg, peach kernel, roasted malt, spina date seed, boat-fruited sterculia seed, stiff silkworm, betel nut and lotus seed) is adopted, total aflatoxin standard substances with different concentrations (15 mug/kg, 10 mug/kg and 2 mug/kg) are manually added, the aflatoxin-negative traditional Chinese medicine sample is obtained after being treated by a quick-test matched pretreatment method (as described in the sixth embodiment), each sample is repeatedly measured for 6-8 times, the detection average value is taken as a detection result, CV and recovery rate are calculated, and the results are shown in tables 1, 2 and 3.

TABLE 1 detection results of the fluorescence immunochromatography detection card on several samples of Chinese medicinal materials (concentration 15. mu.g/kg)

TABLE 2 detection results of the fluorescence immunochromatography detection card on several samples of Chinese medicinal materials (concentration 10. mu.g/kg)

TABLE 3 detection results of the fluorescence immunochromatography detection card on several samples of Chinese medicinal materials (concentration 2. mu.g/kg)

The sensitivity of the detection product is 97.5 percent and the specificity is 100 percent by calculation according to 15 mu g/kg and 2 mu g/kg group references.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The cell strain secreting four subtype antibodies against aflatoxin is characterized by being classified and named as an anti-aflatoxin hybridoma cell strain 8B11 with the preservation number of CCTCC NO: c2019314, the preservation date is 2019, 12 months and 4 days, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university, Wuhan City, China.

2. The antibody secreted by the cell strain secreting the anti-aflatoxin four subtype antibody of claim 1, which is prepared by the following steps:

step two, construction of hybridoma cell strains: myeloma cells and splenocytes are mixed according to a certain proportion, then preheated fusion agent is added at room temperature for fusion, and the fused cells are placed in an incubator for culture after being processed, so as to obtain the cell strains secreting four subtype antibodies against aflatoxin;

step four, producing the antibody: inoculating the hybridoma cell strain to the experimental animal injected with the pristane, and extracting ascites containing the antibody from the abdominal cavity after 8-12 days of inoculation;

and step five, purifying the antibody.

3. The antibody of claim 2, wherein the aflatoxin B1 hapten is injected at a dose of 0.1 mg/dose/individual in step one.

4. The antibody of claim 2, wherein in step two the myeloma cells are in log phase growth.

5. The antibody of claim 2, wherein the ratio of myeloma cells to spleen cells in step two is 1:10 or 1: 5.

6. The antibody of claim 2, wherein the fusion agent in step two is a 45% polyethylene glycol solution containing 5% DMSO, and the fusion time is 60-120 s.

7. The antibody according to claim 2, wherein the antibody is purified by the following steps:

8. An immunochromatography detection card is characterized by being used for rapidly detecting aflatoxin of a traditional Chinese medicine, and the specific preparation method comprises the following steps:

activating fluorescent microspheres by using an activating agent, then adding the antibody or rabbit IgG of claims 2-3, and then oscillating, centrifuging and sealing to obtain an antibody-coupled fluorescent microsphere or rabbit IgG-coupled fluorescent microsphere solution;

and fifthly, drying the combination pad and the NC membrane, then sequentially pasting the NC membrane, the combination pad, the sample pad and the absorbent paper on a bottom plate, and cutting the mixture into test strips with certain widths, namely the immunochromatography detection card.

9. The immunochromatographic test card of claim 8, wherein the activator in step one comprises a solution of the following concentrations: 50mg/ml Sulfo-NHS solution and 50mg/ml EDC solution or 50mg/ml EDC.

10. The immunochromatographic detection card of claim 8, wherein the conjugate diluent in step two is prepared by: after 0.7g Tx-100, 180ml water, 0.242g Tris, 10g sucrose and 2g BSA were completely dissolved, the pH was adjusted to 7.4, water was added to 200ml, filtered and stored at 4 ℃.

11. The immunochromatographic detection card of claim 8, wherein the pretreatment solution in the second step is prepared by: after 0.6057g Tri, 0.5g BSA, 1g Tween 20, 0.073g EDTA and 20ul Proclin 300 were completely dissolved, the pH was adjusted to 7.4, water was added to 100ml, and the solution was stored at 4 ℃.

12. The immunochromatographic detection card of claim 8, wherein the traditional Chinese medicine needs to be pretreated before the immunochromatographic detection card is used for detecting aflatoxin in the traditional Chinese medicine, and the specific treatment steps are as follows:

(1) accurately weighing 15g of medicinal material powder to be detected, adding 40-60ml of 70% methanol solution, and stirring at high speed for 1-2 minutes by using a homogenizer;

13. The immunochromatographic test card of claim 12, wherein the volume ratio of the supernatant or filtrate to the phosphate buffer is 100: 200-800.