CN113754568B - Acid orange I hapten, artificial antigen, synthesis and application thereof - Google Patents

Acid orange I hapten, artificial antigen, synthesis and application thereof Download PDF

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CN113754568B
CN113754568B CN202111109023.2A CN202111109023A CN113754568B CN 113754568 B CN113754568 B CN 113754568B CN 202111109023 A CN202111109023 A CN 202111109023A CN 113754568 B CN113754568 B CN 113754568B
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acid orange
acid
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hapten
artificial antigen
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胥传来
李小玲
匡华
徐丽广
孙茂忠
刘丽强
吴晓玲
宋珊珊
胡拥明
郝昌龙
高巍
马伟
吴爱红
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Jiangnan University
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Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to an acid orange I hapten, an artificial antigen, and synthesis and application thereof. The hapten and the artificial antigen of the acid orange I are successfully synthesized, and the synthesis steps are simple and effective; the monoclonal antibody secreted by the cell strain SLS has better specificity and detection sensitivity on acid orange I, and can realize the detection of acid orange I residues in a marinated product.

Description

Acid orange I hapten, artificial antigen, synthesis and application thereof
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to an acid orange I hapten, an artificial antigen, and synthesis and application thereof.
Background
Acid Orange I (acid Orange I), chemical name of which is 4- [ (4-hydroxy-1-naphthyl) azo ] benzenesulfonic acid monosodium salt, and Orange yellow I, is an artificially synthesized azo dye, and can be used for dyeing acid-base indicators and leather, paper, textiles and the like. Researches prove that azo pigments contain compounds with azo bond structures, naphthalene rings or oxa-anthracene structures, and in the in-vivo biotransformation process, amino compounds are generated, so that the azo pigments have carcinogenicity, damage the subcellular structures of human bodies, interfere the normal functions of various active enzymes, and cause symptoms such as abdominal distension, abdominal pain, dyspepsia and the like. The acid orange I has bright color, stable coloring and low price, and the phenomenon of illegally adding the acid orange I in food production and processing causes serious harm to the health of consumers. The national ministry of health clearly stipulates that synthetic pigments such as acid orange and the like are listed as inedible substances and food additives which are easy to abuse, and the addition of the synthetic pigments into food is prohibited to achieve a coloring effect. Therefore, the method has important significance for the research of the detection method of the acid orange I.
The detection of artificial dyes in recent years has been mainly colorimetric and instrumental. An enzyme-linked immunosorbent assay (ELISA) is a high-efficiency, sensitive and rapid detection method and is increasingly applied to food safety detection. The preparation of monoclonal antibodies of high affinity and high specificity is a prerequisite for immunological detection, where the synthesis of artificial antigens is an important step.
Disclosure of Invention
The invention aims to provide a hapten and an artificial antigen of acid orange I and synthesis thereof, and discloses a hybridoma cell strain SLS of a monoclonal antibody of acid orange I.
According to the technical scheme of the invention, the structural formula of the acid orange I hapten is as follows,
Figure BDA0003273422100000021
compared with the hapten derived from acid orange II in the prior art, the exposed sites of the active structure in the hapten structure of the acid orange I are different.
The second aspect of the invention provides a method for synthesizing the above acid orange I hapten, which is prepared by reacting sulfanilic acid with 1-hydroxy-2-naphthoic acid.
Further, the method comprises the following steps of,
s1: dissolving sulfanilic acid in organic solvent I, adding NaNO under acidic condition2Reacting to obtain solution A;
dissolving 1-hydroxy-2-naphthoic acid in an alkaline solution or an organic solvent II to obtain a solution B;
s2: and adding the solution A into the solution B, adjusting the pH to 8-9, stirring for reaction, and adjusting the pH to acidity to obtain the acid orange I hapten (named AOI).
Specifically, the synthetic route of the hapten AOI is as follows:
Figure BDA0003273422100000022
the derivation process of the hapten of the acid orange I is simpler, the structure of the obtained hapten is not added with more groups compared with the structure of the acid orange I, only one active group of carboxyl (-COOH) is added on the structure of the acid orange I to be used for coupling protein, and the structure of the complete acid orange I can be exposed. The artificial antigen obtained by decoupling protein by utilizing active sites on the hapten and the corresponding monoclonal antibody have strong specificity to acid orange I and low IC 50.
Further, in step S1, the acidic condition may be achieved by adding hydrochloric acid or dilute sulfuric acid.
Further, the molar ratio of the sulfanilic acid to the 1-hydroxy-2-naphthoic acid is 1: 0.9-1.1; preferably 1: 1.
further, the organic solvent I is N, N-Dimethylformamide (DMF) and/or dimethyl sulfoxide (DMSO); the alkaline solution is selected from Na2CO3Solution, NaHCO3One or more of a solution and a NaOH solution; the organic solvent II is ethanol and/or methanol.
Specifically, in the step S2, the solution a is added to the solution B, the pH is adjusted to 8 to 9, the solution is stirred at 4 ℃ for 1.5 to 3 hours (preferably 2 hours) to obtain a red precipitate, the pH of the solution is adjusted to 2.0 to 4.0 (preferably 3.0) with hydrochloric acid, the solution is stirred at 4 ℃ for 20 to 50 minutes (preferably 30 minutes), the red residue is obtained by filtration under reduced pressure, and the crude sample is obtained by drying at 35 to 50 ℃ (preferably 37 ℃);
purifying a crude sample by adopting a recrystallization method, dissolving the crude sample (3.2g) in methanol, standing overnight at room temperature after complete dissolution to separate out a red precipitate, filtering by using filter paper, washing by using petroleum ether for 2-3 times, and drying at 50 ℃ to obtain a red precipitate (2.4g), namely the hapten AOI, wherein the yield is as follows: 75.0 percent.
In a third aspect, the invention provides an acid orange I artificial antigen, which is obtained by coupling the acid orange I hapten and Bovine Serum Albumin (BSA) or chicken Ovalbumin (OVA).
Further, the synthetic method of the acid orange I artificial antigen comprises the following steps:
SS 1: dissolving the hapten AOI, 1-ethyl carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) in DMF and/or DMSO to obtain a solution I;
dissolving BSA or OVA in a buffer solution to obtain a solution II;
SS 2: and adding the solution I into the solution II, adjusting the pH value to 8-9, and reacting to obtain the acid orange I artificial antigen.
Specifically, when BSA is adopted in the step SS1, the obtained acid orange I artificial antigen is AOI-EDC-BSA; when OVA is adopted in the step SS1, the obtained acid orange I artificial antigen is AOI-EDC-OVA.
Further, the molar ratio of the hapten AOI, the hapten NHS and the hapten EDC is 1: 2-4: 2-4; preferably, the ratio is 1: 3: 3.
further, in step SS1, the pH of solution I may be adjusted to acidity by adding 0.01 mol/L2-morpholinoethanesulfonic acid (MES).
Further, the buffer solution is a carbonate buffer solution or a borate buffer solution.
Further, the molar ratio of the BSA or OVA to the hapten AOI is 1: 30-90.
Further, in the step SS2, the solution I is added into the solution II, the pH value is adjusted to 8-9, the reaction is carried out overnight at room temperature (25 +/-5 ℃) to obtain a conjugate, and the conjugate is dialyzed to obtain the acid orange I artificial antigen.
Specifically, the specific operation of the dialysis is as follows: the conjugate was dialyzed in phosphate buffered saline (PBS, 0.01mol/L) every 8h for 3 days in dialysis bags, removed and stored at-20 ℃.
The fourth aspect of the invention provides a hybridoma cell strain SLS secreting acid orange I monoclonal antibody, which is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen, Xilu, No. 1, of the Chaojing city, and is classified and named as monoclonal cell strain, the preservation date is 2021 year, 5 months and 13 days, and the preservation number is CGMCC No. 22326.
Specifically, the preparation method of the hybridoma cell strain SLS comprises the following steps:
1. preparing acid orange I artificial antigen (complete antigen of acid orange I) according to the method;
2. immunization of mice: mixing and emulsifying the complete antigen of acid orange I and an equal amount of complete Freund's adjuvant in an amount of injecting 100 mug/mouse for the first immunization, and injecting BALB/c mouse subcutaneously and multiply through the back of the neck; 4 weeks later, booster immunizations were performed, and the dose of complete antigen was halved (50. mu.g/mouse)Mixing and emulsifying with incomplete Freund's adjuvant, and then performing multiple booster immunizations for 3 weeks; the dose was halved (25. mu.g/mouse) in the case of the boost immunization, and the complete antigen was diluted with physiological saline and injected intraperitoneally into mice. The third immunization of the mouse can be followed by tail-breaking blood sampling detection, and the titer and IC of the mouse serum are detected by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA)50High potency, IC50Low mice were fused;
3. cell fusion and cell line establishment: the mouse spleen cells and SP2/0 tumor cells were fused by the polyethylene glycol (PEG 4000) method, hybridoma cells were selected by using a selective medium (HAT medium), and cell culture was performed using HT medium. After one week of fusion, detecting the positive cell holes and the inhibition effect of the positive cell holes by using an ic-ELISA method, subcloning the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. Carrying out subcloning for four times according to the method to obtain a monoclonal hybridoma cell strain SLS of the acid orange I high-secretion specific antibody;
4. and (3) identification of the properties of hybridoma cell strains: sensitivity and specificity were determined by ic-ELISA.
The fifth aspect of the invention provides an acid orange I monoclonal antibody, which is secreted and generated by the acid orange I monoclonal antibody hybridoma cell strain SLS with the preservation number of CGMCC No. 22326.
The sixth aspect of the invention provides the application of the acid orange I monoclonal antibody in acid orange I detection, which is used for analyzing and detecting residual acid orange I in a marinated product.
Compared with the prior art, the technical scheme of the invention has the following advantages: the invention successfully synthesizes the artificial antigen of the acid orange I, has simple and effective synthesis steps, can be completely used in immunoassay, and provides a convenient way for the research of people in the future; the monoclonal antibody secreted by the cell strain SLS has better specificity and detection sensitivity (IC) on acid orange I50The value is 0.82ng/mL), an immunodetection method and raw materials can be provided for detecting acid orange I residues in a halogen product, and the method has practical application value.
Biological material sample preservation: an acid orange I monoclonal antibody hybridoma cell strain SLS which is preserved in China general microbiological culture Collection center (CGMCC) for short and has the address as follows: the classification of No. 3 Xilu-1 of the Chaozhou area, Beijing, the institute of microbiology of the Chinese academy of sciences is named as monoclonal cell strain, the preservation date is 2021 year, 5 months and 13 days, and the preservation number is CGMCC No. 22326.
Drawings
FIG. 1 is a diagram of ultraviolet identification of immunogen of AOI-EDC-BSA artificial antigen.
FIG. 2 Standard inhibition Curve for monoclonal antibody SLS against acid orange I.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
EXAMPLE 1 Synthesis of hapten AOI
The synthetic route is as follows:
Figure BDA0003273422100000061
1.1 dissolving sulfanilic acid (1.73g, 10.0mmol) in 2mL of DMF, adding 3mL of HCl (1mol/L) solution, stirring at room temperature for 15min, and dropwise adding 10% NaNO2And stirring the mixture for 30min at 4 ℃ until the potassium iodide test paper turns blue to obtain solution A.
1.2 weighing 1-hydroxy-2-naphthoic acid (1.88g, 10.0mmol) and adding to saturated 3mL Na2CO3Cooling the solution to 4 ℃ to obtain solution B.
1.3, slowly dripping the solution A into the solution B, adjusting the pH to 8-9, and stirring for 2h at 4 ℃ to obtain a red precipitate; and finally, regulating the pH value of the solution to 3.0 by using hydrochloric acid, continuing stirring for 30min at 4 ℃, filtering under reduced pressure to obtain a red residue, and drying at 37 ℃ to obtain a crude sample.
1.4, purifying the crude sample by adopting a recrystallization method, and dissolving 3.2g of the crude sample in methanol. Standing overnight at room temperature after complete dissolution to precipitate red precipitate, filtering with filter paper, washing with petroleum ether for 2-3 times, and oven drying at 50 deg.C to obtain red precipitate (2.4g), which is hapten AOI, yield: 75.0 percent.
The amount of sulfanilic acid in step 1.1 was adjusted to 0.173g (1.0mmol), the amount of 1-hydroxy-2-naphthoic acid was adjusted to 0.188g (1.0mmol), and the yield was 81.5%.
EXAMPLE 2 preparation of complete antigen
2.1, 1.6mg of the hapten AOI obtained in example 1, 2.5mg of EDC and 1.5mg of NHS were weighed out, dissolved in 300. mu.L of DMF and stirred at room temperature for 4-6h to give solution I. 10.0mg of BSA (1: 30 molar ratio of BSA to AOI) was weighed and added to 3mL of carbonate buffer to obtain solution II. Dropwise adding the solution I into the solution II at room temperature, adjusting the pH of the mixed solution to 8-9 by using 1mol/L NaOH solution, and reacting overnight at room temperature to obtain a conjugate AOI-EDC-BSA; and replacing BSA with OVA to obtain the conjugate AOI-EDC-OVA.
The amount of hapten AOI was adjusted to 3.3mg, EDC 5.1mg and NHS 3.1mg, so that the molar ratio of BSA to AOI was 1: 60, adding a solvent to the mixture; or
The hapten AOI was adjusted to 5.0mg, EDC to 7.7mg and NHS to 4.6mg, so that the molar ratio of BSA to AOI was 1: 90, conjugate AOI-EDC-BSA can also be prepared.
2.2, dialysis: cutting into 8cm dialysis bag, boiling in boiling water for 3min, cooling, and storing in deionized water at 4 deg.C; and (3) putting the conjugate AOI-EDC-BSA/OVA coupling solution into a dialysis bag, dialyzing in 0.01mol/L PBS (phosphate buffer solution), changing the solution once every 8h, dialyzing for 3 days to obtain the complete antigen AOI-EDC-BSA/OVA, taking out, performing characterization on the conjugate by using an ultraviolet spectrophotometry, and storing at-20 ℃.
The ultraviolet identification chart of the immunogen of the complete antigen AOI-EDC-BSA is shown in figure 1.
Example 3 immunization of mice
Mixing and emulsifying the complete antigen of acid orange I and an equal amount of complete Freund's adjuvant in an amount of injecting 100 mug/mouse for the first immunization, and injecting BALB/c mouse subcutaneously and multiply through the back of the neck; 4 weeks later, booster immunizations were performed, and the dose of complete antigen was halved (50. mu.g/mouse)Mixing and emulsifying with incomplete Freund's adjuvant, and then performing multiple booster immunizations for 3 weeks; the dose was halved (25. mu.g/mouse) in the case of the boost immunization, and the complete antigen was diluted with physiological saline and injected intraperitoneally into mice. The third immunization of the mouse can be followed by tail-breaking blood sampling detection, and the titer and IC of the mouse serum are detected by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA)50High potency, IC50Low mice were subjected to fusion.
Example 4 cell fusion and screening
(1) After three days of spurting immunization, cell fusion is carried out according to a conventional PEG 4000 (polyethylene glycol) method, and the specific steps are as follows:
a. collecting SP2/0 tumor cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2Culturing in an incubator; before fusion, SP2/0 tumor cells were required to reach 1-4 x 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion; during fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
b. after killing the mice by the cervical removal method, the mice are immediately put into 75% alcohol for sterilization, soaked for about 5min, the spleen of the mice is taken out by aseptic operation, ground moderately by a syringe rubber head and passed through a 200-mesh cell screen to obtain spleen cell suspension. Collecting into a 50mL sterile centrifuge tube, centrifuging at 1200r/min for 8min, washing splenocytes with RPMI-1640 medium, picking out more beating tissue impurities, and repeating the operation three times. After the last centrifugation, spleen cells are diluted to a certain volume and counted for later use;
c. the fusion process is 7 min: 1min, 1mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2min, and tightly holding the centrifuge tube with two hands; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 1mL of RPMI-1640 culture medium every 30s at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then, the mixture is incubated at 37 ℃ for 5min, centrifuged at 800r/min for 10min, the supernatant is discarded, the cells in the centrifuge tube are lightly tapped, and RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2% 50 XHAT is added thereto, the mixture is added to a 96-well cell plate at 200. mu.L/well, the plate is placed at 37 ℃,5％CO2culturing in an incubator.
(2) Cell screening and cell strain establishment: half-changing the fused cells with HAT medium on day 3 after cell fusion; on day 5, the whole culture medium was changed with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum and 1% 100 × HT; cell supernatants were taken on day 7 for screening. The screening is divided into two steps: firstly, screening positive cell holes by using an ic-ELISA method, secondly, selecting an acid orange I standard substance, and determining the inhibition effect of the positive cells by using the ic-ELISA method. And selecting a cell hole with better inhibition on the acid orange I standard substance, carrying out subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days. And performing subcloning for four times according to the method to finally obtain the acid orange I monoclonal antibody cell strain SLS.
EXAMPLE 5 preparation and characterization of monoclonal antibodies
Preparation of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 2X 10 per mouse after 7 days6Acid orange I hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the caprylic acid-saturated ammonium sulfate method. Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then precipitating IgG type monoclonal antibody with ammonium sulfate solution with equal saturation, centrifuging, discarding supernatant, dissolving with 0.01mol/LPBS solution (pH 7.4), dialyzing, desalting, and storing at-20 deg.C.
The identification of the monoclonal antibody comprises the following steps,
(1) coating: diluting the coated AOI-EDC-OVA with 0.05mol/L (pH 9.6) carbonate buffer solution from 1 μ g/mL by 3 times, reacting at 37 deg.C for 2h at 100 μ L/well;
(2) washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
(3) and (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;
(4) sample adding: diluting antiserum (which is obtained by diluting the antiserum by a corresponding multiple with an antibody diluent after tail-cutting blood collection of a mouse) from 1:1000 by multiple, adding the diluted antiserum into coated holes of each dilution, reacting at the temperature of 100 mu L/hole for 30 min; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
(5) color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
(6) termination and measurement: adding 50 mu L of stop solution into each hole to stop the reaction, and then measuring the OD 450 value of each hole by using an enzyme-labeling instrument;
determination of IC of monoclonal antibody acid orange I by IC-ELISA50Comprises the following steps: 0.82ng/mL, which shows that the kit has good sensitivity to acid orange I and can be used for immunoassay detection of the acid orange I.
Respectively measuring the cross reaction of the acid orange I monoclonal antibody to structural analogues acid orange II, sunset yellow, acid red 88 and Sudan red I, and according to the formula, the cross reaction rate (CR%) is equal to the IC of the substance to be detected50IC of/analogue50X 100%, the cross-reactivity of this monoclonal antibody to acid orange I is 100%. The result shows that the cross reaction rate of the monoclonal antibody SLS to acid orange II, sunset yellow, acid red 88 and Sudan red I is less than 2%, so that the antibody prepared by the invention has no cross reaction to the 4 medicines and has high specificity.
Solution preparation:
carbonate Buffer (CBS): weighing Na2CO31.59 g，NaHCO32.93 g, adding double distilled water to about 800mL, uniformly mixing, carrying out ultrasonic treatment in a water bath at normal temperature until the double distilled water is completely dissolved, and adding the double distilled water to a constant volume of 1000 mL;
borate buffer solution (BB): 0.525g of Na was weighed2B4O7·10H2O and 0.278g H3BO3Adding double distilled water to a constant volume of 500mL, and storing at 4 ℃ for later use;
MES buffer solution: 9.065g of MES. H were weighed2Dissolving the O in double distilled water, and fixing the volume to 500 mL;
phosphate Buffered Saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
PBST: PBS containing 0.05% tween 20;
TMB color development liquid: solution A: na (Na)2HPO4·12H218.43g of O, 9.33g of citric acid and pure water with constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The liquid B is prepared according to the following steps: 1 to obtain the TMB color developing solution which is mixed at present.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (9)

1. An acid orange I artificial antigen is characterized by being obtained by coupling acid orange I hapten and bovine serum albumin or chicken egg albumin, wherein the acid orange I hapten has a structural formula shown in the specification,
Figure 742275DEST_PATH_IMAGE001
。
2. the acid orange I artificial antigen of claim 1, wherein the acid orange I hapten is prepared by reacting sulfanilic acid with 1-hydroxy-2-naphthoic acid.
3. The acid orange I artificial antigen of claim 2, wherein the acid orange I hapten is synthesized by the method comprising the following steps,
s1: dissolving sulfanilic acid in organic solvent I, adding NaNO under acidic condition2Reacting to obtain solution A;
dissolving 1-hydroxy-2-naphthoic acid in an alkaline solution or an organic solvent II to obtain a solution B;
s2: and adding the solution A into the solution B, adjusting the pH to 8-9, stirring for reaction, and adjusting the pH to acidity to obtain the acid orange I hapten.
4. The acid orange I artificial antigen of claim 3, wherein the molar ratio of the sulfanilic acid to the 1-hydroxy-2-naphthoic acid is 1: 0.9-1.1.
5. The method for synthesizing acid orange I artificial antigen as claimed in any one of claims 1 to 4, comprising the following steps:
SS 1: dissolving the acid orange I hapten, 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide and/or dimethyl sulfoxide to obtain a solution I;
dissolving bovine serum albumin or egg white albumin in a buffer solution to obtain a solution II;
SS 2: and adding the solution I into the solution II, adjusting the pH value to 8-9, and reacting to obtain the acid orange I artificial antigen.
6. The method for synthesizing acid orange I artificial antigen according to claim 5, wherein the molar ratio of the acid orange I hapten, 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide is 1: 2-4: 2-4.
7. A hybridoma cell strain SLS secreting acid orange I monoclonal antibody is preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism research institute No. 3, West Lu No. 1 institute, North Cheng, south China, Beijing, with the preservation date of 2021 year, 5 months and 13 days, and the preservation number is CGMCC No. 22326.
8. The acid orange I monoclonal antibody is characterized by being secreted and generated by the acid orange I monoclonal antibody hybridoma cell strain SLS with the preservation number of CGMCC No. 22326.
9. The use of the acid orange I monoclonal antibody of claim 8 in acid orange I detection.
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