CN107513522B - Hybridoma cell strain secreting anti-ochratoxin monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting anti-ochratoxin monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain F-3-3 secreting anti-ochratoxin monoclonal antibody, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14303. The monoclonal antibody secreted by the cell strain and used for resisting ochratoxin has the advantages of high sensitivity, good specificity, good anti-interference performance and 50% inhibition concentration IC of the ochratoxin A50Is 0.990 mug/L, has better anti-interference performance on possible mycotoxin, pesticide, heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like in the traditional Chinese medicine, provides conditions for the immunodetection of ochratoxin A in the traditional Chinese medicine, and has practical application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain secreting anti-ochratoxin monoclonal antibodies and application thereof.
Background
The traditional Chinese medicinal materials are likely to pollute fungi due to the influence of the properties of the traditional Chinese medicinal materials and external factors in the processes of production, harvesting, processing, transportation, storage and the like, and further mildew and pollute mycotoxin. The mildew can not only affect the quality of the traditional Chinese medicinal materials and cause the traditional Chinese medicinal materials to lose the original medicinal value and cause huge economic loss, but also can cause serious damage to the liver, the kidney, the nerves, the hematopoietic system and the like of a patient and even possibly induce cancer in the mildew process. Meanwhile, along with the popularization of the health care concept of the traditional Chinese medicine, more and more traditional Chinese medicinal materials with homology of medicine and food are used for daily diet and health care of people, the market demand is large, if the traditional Chinese medicinal materials are polluted by mycotoxin, the traditional Chinese medicinal materials inevitably harm the health of more people, the economic burden of individuals and medical systems is increased, and the harmony and stability of the society are more likely to be further influenced.
Ochratoxin (ochratoxin) is a secondary metabolite produced by toxigenic strains of aspergillus and penicillium, and comprises 7 compounds with similar structures, such as ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC), ochratoxin D (OTD), methyl ester of OTA, methyl ester and ethyl ester of OTB, and the like, and the compounds are derivatives of isocoumarin linked with L-phenylalanine. The ochratoxin has serious pollution to crops in the global range, wherein the ochratoxin A has the widest distribution in the nature, the highest pollution level and the strongest toxicity, has the effects of teratogenesis, mutagenesis, carcinogenesis and the like, has immunotoxicity, hepatotoxicity and nephrotoxicity, is considered to be related to Barr's endemic nephropathy, and is most closely related to human health. The international agency for research on cancer (IARC) identified it as a class 2B carcinogen in 1993. Because of serious harm of ochratoxin, the ochratoxin attracts general attention of various countries.
The content of OTA is usually very low, so the detection means is more demanding. At present, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and other chromatography methods are used for measuring OTA, and are gradually used for quite special determination and determination of various mycotoxins in combination with mass spectrometry and nuclear magnetic resonance. The methods have accurate detection and low detection limit, but need complex sample pretreatment process, special instruments and professional personnel training, and are greatly limited in practical application.
The immunoassay method, which is an analysis method for detecting various substances using an antigen-antibody specific binding reaction, can make up all of the above disadvantages, and the key to establishing an immunoassay method for small molecule compounds is the ability to produce antibodies with high affinity and high specificity for small molecule compounds.
Disclosure of Invention
The monoclonal antibody prepared by the cell strain has better specificity and higher detection sensitivity to OTA, has better anti-interference performance to mycotoxin, pesticide, heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like which possibly exist in the traditional Chinese medicine, and lays a foundation for establishing an immunological detection method of OTA in the traditional Chinese medicine.
The hybridoma cell strain secreting the anti-ochratoxin monoclonal antibody is named as an ochratoxin monoclonal antibody hybridoma cell strain F-3-3, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.14303 and the preservation date of 2017, 06 and 15 days.
The anti-ochratoxin monoclonal antibody is secreted and generated by an ochratoxin monoclonal antibody hybridoma cell strain F-3-3 with the preservation number of CGMCC No.14303, and is applied to analysis and detection of ochratoxin A in traditional Chinese medicines.
The invention provides a basic preparation process of an F-3-3 cell strain, which comprises the following steps:
(1) synthesis and identification of OTA hapten: taking 0.5g of 8-hydroxy-3-methyl-1-oxobenzopyran-7-carboxylic acid, adding 20mL of dichloromethane for dissolution, taking 0.31g of oxalyl chloride, adding 0.5mL of N, N-Dimethylformamide (DMF) for dissolution and dilution, dropwise adding the solution into dichloromethane solution, stirring at room temperature for 2h, adding 0.52g of 4-nitrophenylalanine, adding 0.3mL of triethylamine, stirring at room temperature for 2h, stopping reaction, adding 20mL of water, shaking and standing, separating out an aqueous phase, concentrating and evaporating an organic phase, applying to a silica gel column, and eluting and separating ethyl acetate/petroleum ether (V/V, 1/5) to obtain 0.87g of nitroochratoxin; taking 0.87g of nitroochratoxin, adding 50mL of ethanol for dissolving, adding 0.2g of palladium-carbon, introducing hydrogen, stirring at room temperature for 4 hours, stopping reaction, filtering, removing the palladium-carbon, removing the ethanol by rotary evaporation to obtain red oily matter, and recrystallizing in 30mL of dichloromethane/petroleum ether (V/V, 1/1) to obtain 0.76g of aminoochratoxin, namely OTA hapten, the yield is 95%, and the nuclear magnetic resonance hydrogen spectrum identification result;
(2) complete antigen synthesis: respectively coupling the OTA hapten with Bovine Serum Albumin (BSA) and Ovalbumin (OVA) to obtain an immune antigen (OTA-BSA) and an envelope antigen (OTA-OVA);
(3) animal immunization: taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of OTA-BSA; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the physiological saline is used for replacing Freund incomplete adjuvant in the last immunization, intraperitoneal injection is adopted, and the injection dosage is the same as the previous times; detecting serum titer by indirect ELISA;
(4) cell fusion and cell line screening: on the third day after the enhancement of the immunity, fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol (PEG4000) method, culturing by complete culture solution, detecting cell strains secreting OTA by using an indirect ELISA method, determining the inhibition effect of the cell strains by using an indirect competitive ELISA method, screening positive cell strains which are best inhibited, carrying out three times of subcloning, and finally obtaining hybridoma cell strains F-3-3;
(5) antibody preparation and purification and characterization: injecting liquid paraffin into Balb/c mice for 6-8 weeks, wherein each mouse is 500 mu L; injecting 0.5mL hybridoma cell CGMCC No.14303 of each mouse into the abdominal cavity after 10 days, collecting ascites from the seventh day, purifying the ascites by an octanoic acid-ammonium sulfate method, analyzing the purification effect by an SDS-PAGE gel electrophoresis method, measuring the titer, subtype and cross reactivity of the obtained monoclonal antibody, and storing at-20 ℃;
(6) the application of the antibody is as follows: the anti-ochratoxin monoclonal antibody secreted by the hybridoma cell strain F-3-3 is used for preparing an ochratoxin A enzyme linked immunosorbent assay kit for analysis and detection of ochratoxin A in traditional Chinese medicines.
The invention has the beneficial effects that:
(1) the hybridoma cell strain F-3-3 provided by the invention can be used for preparing a high-titer ochratoxin-resistant monoclonal antibody, and the titer measured by an ELISA method for anti-ochratoxin mouse ascites antibodies is not less than 20000.
(2) The anti-ochratoxin monoclonal antibody provided by the invention has the advantages of high sensitivity, good specificity, good anti-interference performance and 50% inhibition concentration IC of ochratoxin A500.990 μ g/L, and can be used for treating mycotoxin, pesticide, and pesticide in Chinese medicinal materials,Heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like have better anti-interference performance.
(3) The anti-ochratoxin monoclonal antibody provided by the invention can be applied to determination of ochratoxin A content in traditional Chinese medicines.
Biological material sample preservation: a hybridoma cell strain secreting anti-ochratoxin monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), and the address is as follows: the collection date of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology of Chinese academy of sciences, 6.06.15 days in 2017, and the collection number is CGMCC No. 14303.
Drawings
FIG. 1 scheme for synthesis of ochratoxin A hapten
FIG. 2 Ochratoxin A competition ELISA standard curve
FIG. 3 Ochratoxin A competition ELISA Logit/Log standard curve
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
Example 1: screening of hybridoma cell line F-3-3
1. Hapten synthesis and identification
The synthesis route of OTA hapten is shown in figure 1.
Taking 0.5g of 8-hydroxy-3-methyl-1-oxobenzopyran-7-carboxylic acid, adding 20mL of dichloromethane for dissolution, taking 0.31g of oxalyl chloride, adding 0.5mL of N, N-Dimethylformamide (DMF) for dissolution and dilution, dropwise adding the solution into dichloromethane solution, stirring at room temperature for 2h, adding 0.52g of 4-nitrophenylalanine, adding 0.3mL of triethylamine, stirring at room temperature for 2h, stopping reaction, adding 20mL of water, shaking and standing, separating out an aqueous phase, concentrating and evaporating an organic phase, applying to a silica gel column, and eluting and separating ethyl acetate/petroleum ether (V/V, 1/5) to obtain 0.87g of nitroochratoxin; taking 0.87g of nitroochratoxin, adding 50mL of ethanol for dissolution, adding 0.2g of palladium-carbon, introducing hydrogen, stirring at room temperature for 4 hours, stopping reaction, filtering, removing the palladium-carbon, removing the ethanol by rotary evaporation to obtain red oily matter, and recrystallizing in 30mL of dichloromethane/petroleum ether (V/V, 1/1) to obtain 0.76g of aminoochratoxin, namely OTA hapten with the yield of 95%.
Taking the hapten, performing nuclear magnetic resonance hydrogen spectrum identification,1H-NMR(CDCl3,300MHz)δ：11.0(1H,s,COOH)，8.45(1H,s,NH)，8.01(1H,d,ArH)，7.14(1H,d,ArH)，7.04(2H,d,ArH)，6.58(2H,d,ArH)，6.27(2H,s,NH2)，5.35(1H,s,OH)，4.72(1H,dd,CH)，4.52(1H,dd,CH)，2.85-3.10(4H,dd,CH2)，1.37(3H,s,CH3). In the spectrum, the chemical shift delta-6.27 is the resonance absorption peak of amino hydrogen on the benzene ring, and the existence of the absorption peak proves that the spacer arm coupling is successful and the OTA hapten structure is correct.
2. Complete antigen synthesis
Immune antigen Synthesis-coupling of OTA hapten to BSA
Dissolving OTA hapten 15mg in 1mL of ethanol, diluting with 0.05mol/L carbonate buffer solution, adding 10% glutaraldehyde aqueous solution 0.2mL, and stirring at room temperature for 1h to obtain solution A; taking 50mg of BSA, and adding 0.05mol/L carbonate buffer solution for dissolving to obtain solution B; adding the solution A into the solution B, stirring at room temperature for 4h, adding 2mg sodium borohydride, stirring for 1h, dialyzing and purifying with 0.02mol/L phosphate buffer solution for 3 days, changing the solution 3 times per day to obtain the immune antigen, subpackaging, and storing at-20 ℃ for later use.
Coating antigen synthesis-coupling of OTA hapten and OVA
Taking 8mg of OTA hapten, adding 0.5mL of water, adding 0.2mL of 1mol/L diluted hydrochloric acid, fully stirring to dissolve the OTA hapten, stirring for 20min at 0-5 ℃, taking 0.2mg of sodium nitrite, dissolving in 1mL of water, dropwise adding the dissolved sodium nitrite into the hapten solution, and continuously stirring for 1h at 0-5 ℃ to obtain a hapten activating solution A; dissolving OVA 50mg in 0.1mol/L carbonate buffer solution, stirring and balancing at 0-5 deg.C for 10min, adding solution A, stirring for 2h, dialyzing and purifying with 0.02mol/L phosphate buffer solution for 3 days, changing solution 3 times per day to obtain coated antigen, packaging, and storing at-20 deg.C.
3. Animal immunization
Taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of OTA-BSA; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.
Table 1 mouse immunization procedure
And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:
(1) diluting the coated antigen (OTA-OVA) with 0.05mol/L of carbonate buffer solution with pH9.6 at a ratio of 1:1000, coating an enzyme label plate with 100 μ L of each well, incubating at 37 ℃ for 2h, removing the coating solution, washing with PBST for 1 time, and patting to dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;
(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of enzyme-labeled secondary antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST, and patting dry at intervals of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) measuring OD value with wavelength of 450nm by enzyme-labeling instrument, and determining OD of sample hole450The titer of positive sera was determined as a dilution factor close to 1.
4. Cell fusion
(1) Preparing feeder cells: the Balb/c mice of 8-10 weeks old are killed after neck breakage, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench with the abdomen facing upwards in a plate or fixed on a dissecting plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.
(2) Preparation of splenocytes: 3d after enhancing the immunity, taking an immune Balb/c mouse, collecting blood from an orbit, dislocating and killing the mouse, disinfecting the mouse in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring the spleen cell suspension into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging the spleen cell suspension at 1500-2000 r/min for 5min, removing a supernatant, adding RPMI-1640 to 30mL, and counting the number for later use.
(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.
(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.
5. Cell line selection
And (3) when the cells grow to 1/2-1/3 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, OTA is selected as a standard substance, and inhibition effect determination is carried out on positive cells by indirect competition ELISA. Wells with better inhibition of OTA standards were selected, subcloned by limiting dilution, and tested in the same manner. Repeating the steps for three times to obtain the cell strain F-3-3 capable of stably secreting the anti-ochratoxin monoclonal antibody. The cell strain has been deposited in China general microbiological culture Collection center (CGMCC for short, the address: the King Kogyo Datun way in Beijing, China academy of sciences, microbiological research institute, postal code 100101) in 2017, 15.06.15.7, and the preservation number is CGMCC No. 14303.
Example 2: preparation, purification and characteristic identification of anti-ochratoxin monoclonal antibody
1. Preparation of ascites
Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. Collecting hybridoma cell CGMCC No.14303 in logarithmic growth phase after 10 days with RPMI-1640 basic culture medium, counting with blood counting plate and microscope to cell concentration of 1.0 × 106～1.5×106In the size per mL range. Each mouse is injected with 0.5mL hybridoma cell CGMCC No.14303 into abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.
2. Antibody purification
The monoclonal antibody is purified by adopting an octanoic acid-ammonium sulfate method, and the method comprises the following specific steps:
(1) the ascites was thawed by taking out from a freezer at-20 ℃. The ascites fluid is filtered with double-layered filter paper to primarily remove fat flakes, cell debris and other impurities. Centrifuging at 12000r/min for 15min, taking supernatant, and discarding precipitate. Accurately measuring the ascites volume;
(2) magnetically stirring and uniformly mixing 1 part by volume of ascites and 3 parts by volume of acetate buffer solution, and adjusting the pH value to 4.5-4.8 by using 2mol/L HCl;
(3) slowly adding n-octanoic acid under magnetic stirring, adding 33 μ L of n-octanoic acid into 1mL of ascites, magnetically stirring at room temperature for 30min, and standing at 4 deg.C for 2 hr;
(4) centrifuging at 12000r/min for 5min, collecting supernatant, filtering with double-layer filter paper, and collecting filtrate;
(5) the volume of the filtrate was measured, 1/10 volumes of 0.1mol/L PBS pH7.4 were added, and the pH was adjusted to 7.4 with 2mol/L NaOH (recording the volume of NaOH);
(6) precooling the supernatant in an ice bath, adding solid ammonium sulfate to 0.277g/mL while stirring, adding the solid ammonium sulfate within 30min, and standing at 4 ℃ overnight;
(7) centrifuging at 12000r/min for 15min, and discarding the supernatant. The pellet was dissolved in a volume of 0.01mol/L PBS. Dialyzing with PB for two days, then dialyzing with 0.01mol/L PBS for two days, collecting dialysate, centrifuging at 12000r/min for 15min, collecting supernatant, and storing at-20 deg.C;
(8) the purified antibody was analyzed for purification by SDS-PAGE gel electrophoresis.
3. Antibody titer determination
The antibody titer was determined by indirect ELISA method, as described in reference example 1, 3. determination of serum titer of animal immunization. The result shows that the titer of the anti-ochratoxin monoclonal antibody is more than or equal to 20000.
4. Antibody subtype identification
The subtype of the anti-ochratoxin monoclonal antibody secreted by the hybridoma cell strain F-3-3 is identified to be IgG by using a commercially available SIGMA subtype identification kit1And (4) molding.
5. Antibody cross-reactivity assay
The indirect competitive ELISA method is adopted for determination, and the specific steps are as follows:
(1) diluting the coated antigen (OTA-OVA) with 0.05mol/L of carbonate buffer solution with pH9.6 at a ratio of 1:1000, coating an enzyme label plate with 100 μ L of each well, incubating at 37 ℃ for 2h, removing the coating solution, washing with PBST for 1 time, and patting to dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;
(3) adding 50 mu L of serial diluted ochratoxin A standard working solution (the concentration is 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mu g/L respectively) into each well, then adding 50 mu L of monoclonal antibody diluted by PBS (1: 20000), reacting at 25 ℃ for 30min, then pouring off the reaction solution, washing 3-5 times by PBST (PBST), and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of enzyme-labeled secondary antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST, and patting dry at intervals of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) and measuring the OD value of the wavelength at 450nm by using a microplate reader.
And (3) drawing a standard curve by taking the logarithmic value of the ochratoxin A concentration as an abscissa and taking the percent absorbance value (the percentage of the OD value of each concentration standard substance to the OD value of a hole without the standard substance) as an ordinate. At the same time, the following interfering substances were diluted with standard buffer solutions to the addition values listed in the tables for analysis, and the cross-reactivity (recovery value/addition value × 100%) was calculated from the OD values corresponding to drug color development and the recovery values analyzed by the standard curve, and the results are shown in table 2.
TABLE 2 anti-interference data for other substances commonly found in Chinese medicine
As can be seen from Table 2, mycotoxins, pesticides, heavy metals, adjuvants used in the processing, and contaminating bacteria which may be present in the Chinese medicinal materials do not interfere with the properties of the anti-ochratoxin monoclonal antibody.
Example 3: application of anti-ochratoxin monoclonal antibody
The anti-ochratoxin monoclonal antibody secreted by the hybridoma cell strain F-3-3 is used for preparing an ochratoxin A enzyme linked immunosorbent assay kit for analysis and detection of ochratoxin A in traditional Chinese medicines.
1. Composition of enzyme linked immunosorbent assay kit
(1) An ELISA plate coated with the OTA-OVA conjugate;
(2) anti-ochratoxin monoclonal antibodies;
(3) horse radish peroxidase-labeled goat anti-mouse anti-antibody;
(4) ochratoxin a standard: the concentrations of the standard solution are respectively 0 mug/L, 0.4 mug/L, 0.8 mug/L, 1.6 mug/L, 3.2 mug/L and 6.4 mug/L;
(5) substrate color developing solution: the liquid A is a 2% carbamide peroxide aqueous solution, and the liquid B is a 1% tetramethylbenzidine aqueous solution;
(6) stopping liquid: 2mol/L sulfuric acid solution;
(7) washing liquid: each 1L of the washing solution was prepared as follows: mixing 10mL of Tween-20, 5g of sodium azide and 990mL of phosphate buffer; wherein the concentration of the phosphate buffer solution is 0.02mol/L, and the pH value is 7.4.
2. Preparation of kit Components
(1) Preparation of enzyme label plate coated with OTA-OVA conjugate
Diluting the OTA-OVA conjugate to the optimal working concentration by using a coating buffer solution, coating a 96-hole polystyrene ELISA plate, incubating at 37 ℃ for 2h by using 100 mu L of each hole, throwing off a coating solution, washing by using PBST for 1 time for 30s each time, patting to dry, then adding 150 mu L of confining solution into each hole, incubating at 37 ℃ for 2h, pouring out liquid in the holes, drying, and storing in a vacuum seal manner by using an aluminum film.
Coating buffer solution: 0.05mol/L carbonate buffer solution with the pH value of 9.6;
sealing liquid: each 1L of the confining liquid is prepared according to the following method: mixing 5mL of horse serum, 1g of sodium azide and 30g of casein, dissolving the mixture by using a phosphate buffer solution, and fixing the volume to 1000 mL; wherein the concentration of the phosphate buffer solution is 0.02mol/L, and the pH value is 7.2.
(2) Preparation of horse radish peroxidase-labeled goat anti-mouse anti-antibody
The goat anti-mouse antibody and horseradish peroxidase (HRP) are coupled by a modified sodium periodate method. The traditional sodium periodate method requires that the molar concentration ratio of enzyme to antibody in a reaction system is 4:1, and horseradish peroxidase generates a plurality of sites for binding with the antibody under the strong oxidation effect, so that the activated horseradish peroxidase molecules serve as bridges for connecting the molecules, the enzyme activity of an enzyme marker is reduced, and a plurality of polymers are mixed in the prepared conjugate. To solve this problem, we modified the conventional method, namely:
(1) the blocking process of the amino group is omitted, because the amino group capable of generating self amino group connection is practically few;
(2) the molar concentration ratio of the horseradish peroxidase to the antibody is reduced to 2:1, the improved method is simpler than the traditional method, and the loss of enzyme activity is reduced.
3. Kit detection method
(1) Sample pretreatment
Weighing 5.0g + -0.05 g pulverized Chinese medicinal sample into 50mL polystyrene centrifuge tube, adding 25mL deionized water, shaking vigorously with oscillator for 5min, and centrifuging at room temperature (20-25 deg.C/68-77 ℉) above 3000g for 5 min; after diluting the sample extract with deionized water at a volume ratio of 1:3, 50. mu.L of the sample extract was used for analysis.
(2) Detection with a kit
Adding 20 muL/hole of OTA standard solution/sample solution, 50 muL/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody and 80 muL/hole of anti-ochratoxin monoclonal antibody into a plate hole, slightly oscillating and uniformly mixing, placing a cover plate with a cover plate film in a dark environment at 25 ℃ for reaction for 20min, pouring out liquid in the hole, adding 250 muL of washing solution into each hole, pouring out liquid in the hole after 30s, repeating the operation for washing the plate for 5 times, and patting the plate dry with absorbent paper; adding 50 μ L of substrate color developing solution A and B into each well, mixing by gentle oscillation, covering with cover plate, and reacting at 25 deg.C in dark environment for 10 min; adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument at 450nm, and measuring the absorbance value of each hole.
(3) Calculation of results
Calculating the percent absorbance value: calculating the ratio of the absorbance value of the obtained standard solution or sample solution to the absorbance value of the blank solution according to the following formula:
in the formula: w-percent absorbance value,%;
a is the average absorbance value of the standard solution or the sample solution;
A0average absorbance value of blank solution (standard solution at a concentration of 0. mu.g/L).
Drawing a standard curve: the calculated percent absorbance (%) was plotted on the ordinate and the log of the concentration of the standard solution (log10) was plotted on the abscissa to obtain a standard curve.
Sample result calculation: substituting the percent absorbance value of the sample liquid into a standard curve, and reading out the corresponding concentration of the sample from the standard curve, wherein the content of ochratoxin A in the sample is calculated according to the following formula:
X＝C×n
in the formula: x represents the content of ochratoxin A in the sample, and the content is mu g/kg;
c-the concentration of ochratoxin A in the sample, μ g/L, as determined from a standard curve;
n-sample dilution factor.
The calculations can also be performed using the data processing software of various microplate readers.
Note: and (4) deducting a blank value from the calculated result, and keeping one decimal number in the obtained result.
4. Evaluation of test Effect
(1) Linear relation
The concentrations of the standard solutions were 0, 0.4, 0.8, 1.6, 3.2 and 6.4. mu.g/L, respectively, and the OD values measured are shown in Table 3. The standard curve is plotted with the percent absorbance values (W,%) as ordinate and the log of the concentration of the standard solution (log10) as abscissa (see FIG. 2). By taking logitW as an ordinate and taking a logarithm of the concentration of the standard solution as an abscissa, and converting the attached figure 2, the logitW and the logarithm of the concentration of ochratoxin A show a good linear relation (shown in figure 3) in the concentration range of 0.4 to 6.4 mug/L, and a regression equation Y is-2.890X +0.091, and R is obtained2＝0.9923。
TABLE 3 OD value measurement results of the standard solutions
(2) Limit of sample detection
The method is characterized in that blank malt, peach kernel, spina date seed, boat-fruited sterculia seed, stiff silkworm, centipede, coix seed, scorpion, leech, lotus seed, rangooncreeper fruit, dried orange peel, polygala root, cassia seed, Chinese date, betel nut, earthworm and platycladi seed samples of different sources are respectively used for measurement, and the detection limit is determined by adding 3 times of standard deviation to the measurement average value. The result shows that the detection limit of the 18 matrixes is less than 5 mug/kg, so that the detection limit of the kit to the traditional Chinese medicine sample is determined to be 5 mug/kg.
(3) Accuracy (recovery) and precision (repeatability)
Taking malt, peach kernel, spina date seed, sterculia seed, stiff silkworm, centipede, coix seed, scorpion, leech, lotus seed, rangooncreeper fruit, dried orange peel, polygala root, cassia seed, Chinese date, betelnut, earthworm and platycladi seed which do not contain OTA as blank sample matrixes, carrying out addition recovery tests of three concentration levels (5 mug/kg, 10 mug/kg and 20 mug/kg), respectively measuring by using three batches of kits, and calculating the sample addition recovery rate and the intra-batch and inter-batch variation coefficients. The results show that the adding recovery rate of 18 matrixes is 70-110%, and the variation coefficient in batch and between batches is less than 15%.
(4) Shelf life of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and OTA addition recovery rate of the kit are within a normal range after 12 months of determination. Considering that abnormal storage conditions appear in the transportation and use processes, the kit is placed at 37 ℃ for 8 days for an accelerated aging test, and as a result, all indexes of the kit completely meet the requirements; considering the occurrence of the freezing condition of the kit, the kit is placed in a refrigerator at the temperature of-20 ℃ for 8 days, and as a result, all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
Claims (3)
1. A hybridoma cell strain secreting anti-ochratoxin monoclonal antibodies is named as ochratoxin monoclonal antibody hybridoma cell strain F-3-3, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.14303 and the preservation date of 2017, 06 and 15 days, and is characterized in that: the hybridoma cell strain is obtained by immunizing a mouse and screening after an immune antigen is obtained by coupling an OTA hapten and bovine serum albumin; the synthesis method of the OTA hapten comprises the following steps: taking 0.5g of 8-hydroxy-3-methyl-1-oxobenzopyran-7-carboxylic acid, adding 20mL of dichloromethane for dissolution, taking 0.31g of oxalyl chloride, adding 0.5mL of N, N-dimethylformamide for dissolution and dilution, dropwise adding the solution into dichloromethane solution, stirring at room temperature for 2h, adding 0.52g of 4-nitrophenylalanine, adding 0.3mL of triethylamine, stirring at room temperature for 2h, stopping reaction, adding 20mL of water, shaking and standing, separating out a water phase, concentrating and evaporating an organic phase, applying to a silica gel column, eluting and separating by ethyl acetate/petroleum ether with a volume ratio of 1/5 to obtain 0.87g of nitroochratoxin; taking 0.87g of nitroochratoxin, adding 50mL of ethanol for dissolving, adding 0.2g of palladium-carbon, introducing hydrogen, stirring at room temperature for 4 hours, stopping reaction, filtering, removing the palladium-carbon, and removing the ethanol by rotary evaporation to obtain red oily matter, and recrystallizing with 30mL of dichloromethane/petroleum ether with the volume ratio of 1/1 to obtain 0.76g of aminoochratoxin, namely the OTA hapten.
2. An anti-ochratoxin monoclonal antibody, which is characterized in that: the ochratoxin monoclonal antibody hybridoma cell strain F-3-3 is secreted and produced by the ochratoxin monoclonal antibody hybridoma cell strain F-3-3 with the preservation number of CGMCC No.14303 in claim 1.
3. The use of an anti-ochratoxin monoclonal antibody as claimed in claim 2, which is characterized in that: the method is used for analyzing and detecting ochratoxin A in the traditional Chinese medicine.
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