CN113582923B - Quinaldine hapten, artificial antigen and antibody as well as preparation methods and application thereof - Google Patents

Quinaldine hapten, artificial antigen and antibody as well as preparation methods and application thereof Download PDF

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CN113582923B
CN113582923B CN202110957176.6A CN202110957176A CN113582923B CN 113582923 B CN113582923 B CN 113582923B CN 202110957176 A CN202110957176 A CN 202110957176A CN 113582923 B CN113582923 B CN 113582923B
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quinaldine
hapten
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罗林
梁洪玮
徐振林
孙远明
沈玉栋
肖治理
王弘
杨金易
雷红涛
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Abstract

The invention provides a quinaldine hapten, an artificial antigen and an antibody as well as preparation methods and application thereof, wherein the hapten 1 is prepared, and the artificial antigen 1 is prepared by utilizing the hapten 1; the 4-amino quinaldine is used as hapten 2, hapten 2 is used for coupling carrier protein to obtain artificial antigen 2, a specific antibody for detecting the quinaldine is further prepared, artificial antigen 1 is used as a coating antigen, the antibody has good sensitivity and specificity to the quinaldine, the half inhibition concentration is 1.78 mug/mL, the minimum detection limit is 0.19 mug/mL, the cross reaction rate to common fish anesthetic is lower than 1.32%, an immunoassay method of the quinaldine is established, and the purpose of rapidly and accurately detecting the quinaldine in the fish anesthetic is realized.

Description

Quinaldine hapten, artificial antigen and antibody as well as preparation methods and application thereof
Technical Field
The invention relates to the technical field of food safety detection, in particular to a quinaldine hapten, an artificial antigen and an antibody, and a preparation method and application thereof.
Background
Quinaldine is a quinoline compound which is colorless oily liquid and is also called dimethylquinoline. It has antibacterial and antipyretic properties and is widely used as a parent compound for the preparation of medicaments, especially antimalarial medicaments, as well as bactericides and biocides. Quinaldine can be used for short-time anesthesia operation of fish, and is helpful for fish transportation.
However, if the food is taken with excessive amounts of quinaldine, the health of the human is seriously threatened. The approval use and management of the fishing anesthetic in China is not clearly defined, and in order to prevent the misuse of the quinaldine on the aquatic products, the establishment of a detection method of the quinaldine anesthetic in the aquatic products is urgent.
Currently, there are few methods for detecting quinaldine, mainly ultraviolet spectrophotometry (Hunn J, allen J.Gen Pharmacol-Vasc S,1975,6 (1): 15), LC-MS/MS (Zheng Xianghua, sun Ting, chen Yan, lin Jianzhong, xu Duiming, lily. Solid-phase extraction-liquid chromatography tandem mass spectrometry is used for simultaneously measuring the residual amount [ J ]. Food science and technology, 2020,45 (04): 333-337 ] of 3 kinds of anesthetic for fish in aquatic products, high performance liquid chromatography (Xu Shanlang, yellow and, high level, chen Rimeng, zeng Dandan, chen Yingshou. High performance liquid chromatography is used for measuring the residual amount [ J ]. Analytical laboratory, 6210352 (09): 1035-9.) of quinaldine anesthetic in aquatic products, and the like. The detection limit of the method is mainly 0.2-15 mug/kg, and the ultraviolet spectrophotometry is simple to operate, but has low sensitivity and insufficient anti-interference, and is difficult to meet the requirement of quinaldine trace analysis in aquatic products; the LC-MS/MS method has high sensitivity and accurate quantification, but the instrument and equipment are expensive, the popularization rate is low, the method is difficult to popularize and use in primary line detection, and the method is not suitable for carrying out rapid screening on a large number of sample sites, so that a rapid and simple detection method for quinaldine needs to be invented. Furthermore, there is currently no report on quinaldine hapten, antigen and antibody that can be used for immunoassays.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of a quinaldine detection method in the prior art and provides a quinaldine hapten, an artificial antigen and an antibody as well as a preparation method and application thereof.
The invention aims to provide a quinaldine hapten.
The invention also aims to provide application of the quinaldine hapten in preparation of quinaldine artificial antigen.
The invention also aims at providing a quinaldine artificial antigen: artificial antigen 1 or artificial antigen 2.
The invention also aims to provide application of the quinaldine hapten and/or the artificial antigen in preparation of quinaldine artificial antibodies.
The invention also aims to provide an artificial antigen combination of quinaldine.
The invention also aims to provide a quinaldine antibody.
The invention also aims to provide a kit for detecting quinaldine.
The invention also aims at providing an immunoassay method for detecting quinaldine.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a quinaldine hapten 1, the structural formula of the hapten 1 is shown as a formula (I),
Figure BDA0003220780180000021
hapten 1 was named 2-methylquinoline-4-aminobutyric acid using the systematic nomenclature.
The preparation method of the hapten 1 comprises the following steps:
dissolving 4-aminoquinaldine and ethyl 4-bromobutyrate in N, N-dimethylformamide, adding K 2 CO 3 Stirring and refluxing at 70 ℃ for reaction overnight, extracting with ethyl acetate, drying, adding lithium hydroxide and primary water for hydrolysis, extracting with ethyl acetate, adjusting acid in water phase, extracting with n-butanol, and drying to obtain quinaldine hapten.
Preferably, the 4-aminoquinaldine: ethyl 4-bromobutyrate: k (K) 2 CO 3 Molar ratio = 1-2: 2-3: 1 to 2.
Further preferably, the 4-aminoquinaldine: ethyl 4-bromobutyrate: k (K) 2 CO 3 Molar ratio = 1:2:1.45.
preferably, the 4-aminoquinaldine: molar ratio of lithium hydroxide: primary water=1 to 2: 3-4: 50 to 60.
Further preferably, the 4-aminoquinaldine: molar ratio of lithium hydroxide: primary water = 1:3.81:55.55.
preferably, the pH is adjusted to 4-5.
Further preferably, the acid adjustment is to adjust the pH to 5.
The application of the hapten 1 in preparing quinaldine artificial antigen is also within the protection scope of the invention.
Application of 4-amino quinaldine as hapten 2 in preparation of quinaldine artificial antigen. As known, the structural formula of the 4-amino quinaldine is shown as a formula (II), and the research of the invention shows that the 4-amino quinaldine can be used as hapten to prepare quinaldine artificial antigen;
Figure BDA0003220780180000031
a quinaldine artificial antigen comprises an artificial antigen 1 or an artificial antigen 2, wherein the structural formula of the artificial antigen 1 is shown as a formula (III),
Figure BDA0003220780180000032
the structural formula of the artificial antigen 2 is shown as a formula (IV),
Figure BDA0003220780180000033
preferably, the carrier protein (protein) is Bovine Serum Albumin (BSA), lactoferrin (LF) or chicken Ovalbumin (OVA).
According to the preparation method of the artificial antigen 1, hapten 1 is utilized to couple carrier protein through an active ester method.
As a specific embodiment of the above method, the method for preparing artificial antigen 1 comprises the following steps:
dissolving carrier protein in CBS to obtain carrier protein solution; dissolving hapten 1 in MES water solution, adding EDC and NHS, and stirring at room temperature for reaction in a dark place to obtain hapten 1 activating solution; adding the carrier protein solution into the hapten 1 activating solution in ice bath, uniformly stirring, and coupling overnight at room temperature in a dark place; dialyzing the coupling mixture at low temperature for several days to obtain the artificial antigen 1.
Preferably, the preparation method of the artificial antigen 1 comprises the following steps:
(1) Dissolving a carrier protein in CBS with ph=9.6 to obtain a carrier protein solution;
(2) Dissolving hapten 1 in 0.1M MES water solution to obtain hapten 1 solution;
(3) EDC and NHS are added into the hapten 1 solution in the step (2) to react for 4 hours at room temperature in a dark place, so as to obtain active ester; the molar ratio of EDC to NHS to hapten 1 is 1-2: 1-2: 1, a step of;
(4) Dripping the active ester in the step (3) into the carrier protein solution in the step (1), and coupling overnight at room temperature in a dark place; dialyzing the coupling mixture for 3 days at 4 ℃ with PBS buffer solution to obtain an artificial antigen 1; the mol ratio of the hapten 1 to the carrier protein is 30-300: 1.
further preferably, the molar ratio of EDC and NHS to hapten 1 is 1.5:1.5:1.
further preferably, the molar ratio of hapten 1 to carrier protein is 300:1.
according to the preparation method of the artificial antigen 2, 4-aminoquinaldine, namely hapten 2, is utilized to couple carrier protein by a glutaraldehyde method.
As a specific embodiment of the above method, the method for preparing artificial antigen 2 comprises the following steps:
dissolving carrier protein in PBS buffer solution, then adding glutaraldehyde solution, stirring at room temperature for reaction, and dialyzing with PBS buffer solution for several hours to obtain protein-glutaraldehyde crosslinking solution; dissolving hapten 2 in methanol, then adding the methanol into protein-glutaraldehyde crosslinking solution, stirring at a low temperature for reaction overnight, adding sodium borohydride aqueous solution after the reaction is finished, and carrying out reduction reaction for 2 hours at room temperature; the conjugate mixture was dialyzed against PBS buffer at low temperature for several days to obtain artificial antigen 2.
Preferably, the preparation method of the artificial antigen 2 comprises the following steps:
(1) Dissolving carrier protein in PBS buffer solution with pH=7.4 to obtain carrier protein solution;
(2) Adding 25% glutaraldehyde solution to the carrier protein solution of step (1); the volume ratio of glutaraldehyde solution to carrier protein solution is 2-10: 1000, stirring and reacting for 4 hours at room temperature, dialyzing the reaction solution with PBS buffer solution for 3 times and 3 hours at one time to obtain protein-glutaraldehyde crosslinking solution;
(3) Dissolving hapten 2 in methanol, then adding the methanol into the protein-glutaraldehyde crosslinking solution in the step (2), and stirring and reacting at 4 ℃ for overnight; the mol ratio of hapten 2 to carrier protein is 30-300: 1.
(4) 1mg/mL sodium borohydride aqueous solution is taken and added into the reaction liquid in the step (3) in a dropwise manner; the mass ratio of the sodium borohydride to the carrier protein is 1:100, carrying out reduction reaction for 2h at room temperature; the reacted coupling mixture was dialyzed against PBS buffer for 3 days at 4℃to obtain artificial antigen 2.
Preferably, the volumetric ratio of glutaraldehyde solution to carrier protein solution is 3.75:1000
Preferably, the molar ratio of hapten 2 to carrier protein is 100:1.
the use of the artificial antigen 1 and/or the artificial antigen 2 in the preparation of quinaldine antibodies is also within the scope of the present invention.
Preferably, the artificial antigen 2 is used for preparing quinaldine antibody.
A quinaldine artificial antigen combination comprising an artificial antigen 1 and an artificial antigen 2.
Preferably, the artificial antigen combination comprises an artificial antigen 2 as an immunogen and an artificial antigen 1 as a coating antigen.
Further preferably, the artificial antigen 2 as an immunogen is an artificial antigen 2 (hapten 2-LF) using a carrier protein as Lactoferrin (LF), and the artificial antigen 1 as a coating antigen is an artificial antigen 1 (hapten 1-OVA) using a carrier protein as chicken Ovalbumin (OVA).
The use of the artificial antigen combination in the preparation of antibodies to quinaldine and/or in the detection of quinaldine is also within the scope of the present invention.
Preferably in a kit for detecting quinaldine or in an immunoassay method.
A quinaldine antibody is prepared by immunizing animals with artificial antigen 1 or artificial antigen 2.
Preferably, the antibodies are prepared by immunizing an animal with artificial antigen 2.
Further preferably, the antibody is prepared by immunizing an animal with an artificial antigen 2 (hapten 2-LF) using a carrier protein as Lactoferrin (LF).
Preferably, the antibody is a polyclonal antibody.
A method for preparing quinaldine antibody, which is to immunize experimental animal by using artificial antigen 2 to make its body produce specific antibody against quinaldine, wherein the antibody is immunoglobulin G which can generate specific immune reaction with quinaldine.
As a specific embodiment of the above method, the preparation method of the quinaldine antibody comprises the following steps:
(1) Immunizing an experimental animal by utilizing the artificial antigen 2 and the Freund's adjuvant;
(2) In the first immunization, the artificial antigen 2 is emulsified by using an equal volume of Freund's complete adjuvant to immunize Balb/c mice of 6-7 weeks old, and the immunization dose of each emulsified mouse is 0.1 mL/mouse;
(3) In the boosting, balb/c mice were immunized after emulsification with the same dose of artificial antigen 2 and an equal volume of Freund's incomplete adjuvant;
(4) After four times of booster immunization, heart blood is taken and separated to obtain serum, namely the mouse polyclonal antibody.
Preferably, the experimental animal is immunized with the artificial antigen 2 (hapten 2-LF) with the carrier protein being Lactoferrin (LF).
The use of quinaldine antibodies in the detection of quinaldine is also within the scope of the present invention.
Preferably, it is the use of quinaldine antibodies in a kit for detecting quinaldine
A kit for detecting quinaldine, comprising a quinaldine artificial antigen as a coating antigen and an antibody prepared by immunizing an animal with the quinaldine artificial antigen.
Preferably, the kit comprises the artificial antigen 1 as a coating antigen and an antibody prepared by immunizing an animal with the artificial antigen 2.
Preferably, the kit comprises an antibody prepared by immunizing an animal with an artificial antigen 1 with a carrier protein as chicken Ovalbumin (OVA) and an artificial antigen 2 with a carrier protein as Lactoferrin (LF) as coating sources.
Preferably, the kit comprises an enzyme-labeled plate coated with the quinaldine artificial antigen serving as a coating source, a quinaldine standard substance, an antibody obtained by immunizing an animal with the quinaldine artificial antigen, an enzyme-labeled secondary antibody working solution, a chromogenic solution, a stop solution, a concentrated diluent and a concentrated washing solution.
Further preferably, the coating antigen is artificial antigen 1; the antibody is obtained by immunizing animals with the artificial antigen 2.
Further preferably, the coating antigen is chicken Ovalbumin (OVA) artificial antigen 1 as carrier protein; the antibody is prepared by immunizing animals with an artificial antigen 2 taking carrier protein as Lactoferrin (LF).
An immunoassay method for detecting quinaldine uses an artificial antigen 1 as a coating antigen and uses an antibody prepared by immunizing an animal with the artificial antigen 2 as an immunogen as a detection antibody for detection.
Preferably, in the immunoassay method, an antibody prepared by immunizing an animal with an artificial antigen 1 (hapten 1-OVA) with a carrier protein being chicken Ovalbumin (OVA) as a coating antigen and an artificial antigen 2 (hapten 2-LF) with a carrier protein being Lactoferrin (LF) as an immunogen is used as a detection antibody.
As a specific embodiment of the method, the method comprises the following steps:
(1) Immunizing an animal with an artificial antigen 2 to prepare a polyclonal antibody;
(2) Coating the artificial antigen 1 as a coating source on a micro-pore plate, and then adding the polyclonal antibody prepared in the step (1) into the micro-pore plate;
(3) And adding a quinaldine sample to be tested, and measuring the content of the quinaldine by adopting competition ELISA.
Preferably, the carrier protein of the artificial antigen 2 is Lactoferrin (LF), i.e. hapten 2-LF.
Preferably, the carrier protein of the artificial antigen 1 is chicken Ovalbumin (OVA), i.e. hapten 1-OVA.
Preferably, the artificial antigen 1 (hapten 1-OVA) conjugated to chicken Ovalbumin (OVA) is coated at a concentration of 0.5 μg/mL with a 2000 fold dilution of quinaldine antibody.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
The hapten 1 is quinaldine hapten 1; the 4-amino quinaldine is hapten 2; the artificial antigen 1 is quinaldine artificial antigen 1; the artificial antigen 2 is quinaldine hapten artificial antigen 2; the antibody is quinaldine antibody.
Compared with the prior art, the invention has the following beneficial effects:
the invention prepares quinaldine hapten 1, and uses hapten 1 to couple carrier protein to obtain artificial antigen 1; the 4-amino quinaldine is used as hapten 2, hapten 2 is used for coupling carrier protein to obtain artificial antigen 2, and further a specific antibody for detecting the quinaldine is prepared, the artificial antigen 1 is used as an artificial coating antigen, the antibody has good sensitivity and specificity to the quinaldine, the half inhibition concentration is 1.78 mug/mL, the minimum detection limit is 0.19 mug/mL, the cross reaction rate to common fish anesthetic is lower than 1.32%, the antibody has better specificity to the quinaldine, the interference of other fish anesthetic can be effectively eliminated, and a core reagent is provided for establishing an immunodetection method of the fish anesthetic quinaldine. The quinaldine hapten, the artificial antigen and the antibody of the invention are utilized to realize the purpose of rapidly and accurately detecting the quinaldine in the fish anesthetic.
Drawings
FIG. 1 is a mass spectrum identification chart of hapten 1 of example 1 of the present application.
FIG. 2 shows ultraviolet scans of hapten 1, artificial antigen 1 (hapten 1-OVA) and OVA in example 2 of the present application.
FIG. 3 shows the ultraviolet scans of hapten 2, artificial antigen 2 (hapten 2-LF) and LF in example 3 of the present application.
FIG. 4 is a graph showing the inhibition of quinaldine by an antibody prepared using artificial antigen 2 (hapten 2-LF) as an immunogen in example 6 of the present application.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 Synthesis and identification of quinaldine hapten 1
1. Synthesis of quinaldine hapten 1
1.58g of 4-aminoquinaldine and 3.86g of ethyl 4-bromobutyrate are dissolved in 10mL of N, N-dimethylformamide, and 2g of K are added 2 CO 3 Reflux-reacting at 70deg.C overnight, extracting with ethyl acetate, collecting organic phase, evaporating to dryness, adding 1.6g lithium hydroxide and 10mL primary water, stirring until clear, adding separating funnel, extracting with ethyl acetate, collecting water phase, adding concentrated hydrochloric acid to adjust pH to 5, extracting with n-butanol, collecting organic phase, evaporating to dryness, and collecting brown powder.
2. Identification of quinaldine hapten 1
Results of ESI-MS identification of hapten 1: as shown in fig. 1, as can be seen from fig. 1, MS of hapten 1: c (C) 16 H 15 NO 4 ：285.10，ESI-[M-H]-:：283.90。
The structural formula of the hapten 1 is shown as a formula (I):
Figure BDA0003220780180000081
hapten 1 was named 2-methylquinoline-4-aminobutyric acid using the systematic nomenclature.
EXAMPLE 2 Synthesis and identification of quinaldine Artificial antigen 1
1. Synthesis of quinaldine artificial antigen 1
Hapten 1 prepared in example 1 was conjugated to Lactoferrin (LF) and Ovalbumin (OVA) by the active ester method; the lactoferrin is carrier protein of immune antigen, and the chicken ovalbumin is carrier protein of coating antigen.
Dissolving 32.53mg of hapten 1 prepared in example 1 in 0.1M 2- (N-maline) ethanesulfonic acid (MES) water solution, stirring, adding 7.3mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 4.3mg of N-hydroxysuccinimide (NHS), and stirring at room temperature for reacting for 4 hours in a dark place to obtain hapten 1 activating solution; dissolving 20mg of Lactoferrin (LF) and 20mg of OVA in 5mL of Carbonate Buffer Solution (CBS) of PH9.6 respectively, stirring in an ice bath, adding hapten 1 activating solution, stirring uniformly, and coupling overnight at room temperature in a dark place; dialyzing the coupling mixture with PBS buffer solution at 4deg.C for 3 days, changing dialysate for 2 times per day to obtain artificial antigen 1 (hapten 1-LF, hapten 1-OVA), subpackaging the artificial antigen 1 at concentration of 1mg/mL, and freezing in a refrigerator at-20deg.C.
2. Identification of quinaldine Artificial antigen 1
The ultraviolet scanning measurement (190-400 nm) is carried out on carrier proteins (LF, OVA), hapten 1-LF and hapten 1-OVA, the measurement results are shown in figure 2, and the figure 2 shows that the ultraviolet absorption peak of the artificial antigen 1 (hapten 1-LF, hapten 1-OVA) has obvious blue shift compared with the ultraviolet absorption peak of the hapten 1, and the artificial antigen 1 simultaneously has the characteristic absorption peaks of the hapten, LF and OVA, which indicates that the coupling of the artificial antigen 1 is successful.
EXAMPLE 3 Synthesis and identification of quinaldine Artificial antigen 2
1. Synthesis of quinaldine artificial antigen 2
The synthesis method of the quinaldine artificial antigen 2 comprises the following steps:
lactoferrin (LF) and chicken Ovalbumin (OVA) were coupled by glutaraldehyde using commercially available 4-aminoquinaldine as hapten 2.
Dissolving 20mg of Lactoferrin (LF) and 20mg of chicken Ovalbumin (OVA) in 4ml of 0.01MPBS buffer solution respectively, stirring, adding 15 mu L of 25% glutaraldehyde solution, stirring at room temperature, reacting for 4 hours, dialyzing the reaction solution with PBS buffer solution for 3 times at 4 ℃, and replacing the dialyzate for 3 hours to obtain protein-glutaraldehyde cross-linking solution; dissolving 20mg of hapten 2 in 600 mu L of 0.1M MES water solution, dripping the solution into protein-glutaraldehyde crosslinking solution (300 mu L is added to LF, 600 mu L is added to OVA), stirring uniformly, and coupling overnight at 4 ℃; 200 mu L of 1mg/mL sodium borohydride aqueous solution is dripped into the reaction solution, and stirred and reduced for 2 hours at room temperature; after the reaction, the coupling mixture is dialyzed for 3 days at 4 ℃ in PBS buffer solution, the dialysate is changed for 2 times per day, and then the artificial antigen 2 (hapten 2-LF and hapten 2-OVA) is obtained, and the artificial antigen 2 is packaged at the concentration of 1mg/mL and frozen in a refrigerator at the temperature of minus 20 ℃.
2. Identification of quinaldine Artificial antigen 2
The ultraviolet scanning measurement (190-400 nm) is carried out on carrier proteins (LF, OVA), hapten 2-LF and hapten 2-OVA, the measurement results are shown in figure 3, the ultraviolet absorption peak of the artificial antigen 2 (hapten 2-LF, hapten 2-OVA) has obvious blue shift compared with the ultraviolet absorption peak of the hapten 2, and the artificial antigen 2 simultaneously has characteristic absorption peaks of the hapten, LF and OVA, which indicates that the coupling of the artificial antigen 2 is successful.
Example 4 preparation and identification of antibodies
1. Preparation of antibodies
The artificial antigen 1 (hapten 1-LF) of the conjugated Lactoferrin (LF) prepared in example 2 and the artificial antigen 2 (hapten 2-LF) of the conjugated Lactoferrin (LF) prepared in example 3 were emulsified uniformly with equal amounts of immunological adjuvants (Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the subsequent booster immunization). Balb/C mice with the size of 6-7 weeks are immunized by adopting various injection modes of subcutaneous back, subcutaneous parts, abdominal cavity and feet respectively, and are immunized for the second time after 2 weeks, and the immunization is enhanced every 2 weeks. The tail of the mice was bled 1 week after the fourth booster immunization and serum titers were determined using an indirect competition ELISA. When the potency no longer increases, intraperitoneal injection is used to boost the immunity. After 3 days, the heart takes blood, water bath is carried out for 0.5 to 1 hour, centrifugation is carried out for 15 minutes at 4 ℃ and 10000, the supernatant is taken as antiserum, and the antiserum is quinaldine polyclonal antibody.
2. Identification of antibodies
The antiserum titer and inhibition rate obtained by immunizing Balb/C mice with the artificial antigen 1 (hapten 1-OVA) of the conjugated chicken Ovalbumin (OVA) prepared in example 2 and the artificial antigen 2 (hapten 2-OVA) of the conjugated chicken Ovalbumin (OVA) prepared in example 3 were used as coating antigen, respectively, and detected by ELISA.
Carbonate buffer (CBS, ph=9.6) as a dilution of the coating antigen, phosphate buffer (PBS, 0.01M, ph=7.4) as a dilution of antisera and standards, and tween phosphate buffer (PBST, 0.01M) as a dilution of horseradish peroxidase-labeled goat anti-mouse solution. The coating antigen was diluted to 1. Mu.g/mL, added to a 96-well ELISA plate, and incubated at 4℃overnight at 100. Mu.L per well. After washing the plates 2 times with PBST, 120. Mu.L of 5% bovine serum albumin was added to each well, incubated in a 37℃water bath for 3 hours, and dried at 37℃for 1 hour after spin-drying. Antisera were diluted 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000-fold, respectively, and quinaldine standard was formulated as a 1 μg/mL solution. The solution is added into a 96-well plate coated with the corresponding coating raw material according to a general indirect competition ELISA method, incubated for 40min in a 37 ℃ water bath box, PBST is washed for 5 times, 100 mu L of goat anti-mouse solution marked by horseradish peroxidase with 5000 times of dilution is added, incubation is carried out for 30min in the 37 ℃ water bath box, PBST is washed for 5 times, 100 mu L of 3,3', 5' -tetramethyl benzidine (TMB) substrate chromogenic solution is added, incubation is carried out for 10min in the 37 ℃ water bath box again, 10% concentrated sulfuric acid is added to terminate the reaction, and data are read on an enzyme-labeled instrument. The results of the indirect competition ELISA assay are shown in table 1.
The antiserum with absorbance (OD) of 1-1.5 at 450nm wavelength is selected as the titer of antiserum, and the inhibition ratio=100- (OD inhibition/OD titer) ×100
TABLE 1 results of indirect competition ELISA assay
Figure BDA0003220780180000111
From table 1, it is clear that the combination of artificial antigens with the optimal potency and inhibition ratio selection effect of the antiserum is optimal in combination with the potency of 4000 and the inhibition ratio of 38.46%, that is, the result of the immunodetection of the antigen with the carrier protein as the artificial antigen 2 of lactoferrin (hapten 2-LF) as the immunogen and the carrier protein as the artificial antigen 1 of chicken ovalbumin (hapten 1-OVA) as the coating antigen is optimal.
Example 5 coating concentration and antibody dilution
The effect of coating concentration and quinaldine polyclonal antibody concentration on quinaldine detection was detected using a checkerboard assay. The quinaldine antibody (Ab-hapten 2-LF) prepared by the artificial antigen 2 (hapten 2-LF) coupled to LF of example 4 was used as an antibody, and the artificial antigen 1 (hapten 1-OVA) coupled to OVA prepared in example 2 was used as a coating antigen.
Carbonate buffer (CBS, ph=9.6) as a dilution of the coating antigen, phosphate buffer (PBS, 0.01M, ph=7.4) as a dilution of the quinaldine antibody and standard, and tween phosphate buffer (PBST, 0.01M) as a dilution of horseradish peroxidase-labeled goat anti-mouse solution. Artificial antigen 1 was diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625. Mu.g/mL, respectively, and added to 96-well ELISA plates and incubated at 4℃overnight at 100. Mu.L per well. After washing the plates 2 times with PBST, 120. Mu.L of 5% bovine serum albumin was added to each well, incubated in a 37℃water bath for 3 hours, and dried at 37℃for 1 hour after spin-drying. The quinaldine antibody is diluted 1000 times, 2000 times, 4000 times, 8000 times, 16000 times, 32000 times and 64000 times respectively, and the quinaldine standard substance is prepared into a solution with the concentration of 1 mug/mL; the above solution was loaded in a 96-well plate coated with the corresponding coating antigen according to a general indirect competition ELISA method, 50. Mu.L of quinaldine antibody and 1. Mu.g/mL of quinaldine standard at each dilution ratio were incubated in a 37℃water bath for 40min, PBST plates were washed 5 times, 100. Mu.L of horseradish peroxidase-labeled goat anti-mouse solution was added in a 5000-fold dilution water bath for 30min, PBST plates were washed 5 times in a 37℃water bath, 100. Mu.L of 3,3', 5' -Tetramethylbenzidine (TMB) substrate color development solution was added, the reaction was stopped by adding 10% concentrated sulfuric acid again in a 37℃water bath, and the absorbance (OD) was measured by an enzyme-labeled analyzer.
The inhibition rate was calculated by selecting the coating concentration and the dilution factor of the antibody at a wavelength of 450nm and the absorbance value of 1 to 1.5, and the detection results are shown in Table 2.
Table 2 chessboard test results
OD value Coating concentration Antibody dilution fold Inhibition rate
1.187 2μg/mL 1:16000 34.67％
1.265 1μg/mL 1:4000 37.86％
1.342 0.5μg/mL 1:2000 43.34％
As is clear from Table 2, the highest inhibition ratio corresponds to a coating antigen concentration of 0.5. Mu.g/mL and a quinaldine polyclonal antibody dilution of 2000.
Example 6 sensitivity of antibodies
An enzyme-linked immunoassay (ELISA) standard curve was drawn with antisera (quinaldine antibodies) at different dilution factors according to the coating concentrations determined in example 5. The quinaldine antibody (Ab-hapten 2-LF) prepared by the artificial antigen 2 (hapten 2-LF) coupled to LF of example 4 was used as an antibody, and the artificial antigen 1 (hapten 1-OVA) coupled to OVA prepared in example 2 was used as a coating antigen.
Carbonate buffer (CBS, ph=9.6) as a dilution of the coating antigen, phosphate buffer (PBS, 0.01M, ph=7.4) as a dilution of the quinaldine antibody and standard, and tween phosphate buffer (PBST, 0.01M) as a dilution of horseradish peroxidase-labeled goat anti-mouse solution. Artificial antigen 1 was diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625. Mu.g/mL, respectively, and added to 96-well ELISA plates and incubated at 4℃overnight at 100. Mu.L per well. After washing the plates 2 times with PBST, 120. Mu.L of 5% bovine serum albumin was added to each well, incubated in a 37℃water bath for 3 hours, and dried at 37℃for 1 hour after spin-drying. The quinaldine antibody is diluted 1000 times, 2000 times, 4000 times, 8000 times, 16000 times, 32000 times and 64000 times respectively, and the quinaldine standard substance is prepared into a solution with the concentration of 1 mug/mL; the above solution was loaded in a 96-well plate coated with the corresponding coating antigen according to a general indirect competition ELISA method, 50. Mu.L of quinaldine antibody and 1. Mu.g/mL of quinaldine standard at each dilution ratio were incubated in a 37℃water bath for 40min, PBST plates were washed 5 times, 100. Mu.L of horseradish peroxidase-labeled goat anti-mouse solution was added in a 5000-fold dilution water bath for 30min, PBST plates were washed 5 times in a 37℃water bath, 100. Mu.L of 3,3', 5' -Tetramethylbenzidine (TMB) substrate color development solution was added, the reaction was stopped by adding 10% concentrated sulfuric acid again in a 37℃water bath, and the absorbance (OD) was measured by an enzyme-labeled analyzer.
Taking an OD value as an ordinate and a corresponding standard concentration logarithmic value as an abscissa, performing curve fitting on the function by applying four parameters of origin8.5 software: y= (a-D)/[ 1+ (X/C) B]+D wherein A and D represent the minimum and maximum absorbance (OD) of the drug concentration, respectively, C is the midpoint concentration, OD when the standard concentration is equal to C is (A+D)/2, just at the inflection point of the curve, half the inhibitory concentration is IC 50 B represents the steepness of the curve and is called a slope factor; by IC 10 For detection limit, IC is used 20 ～IC 80 Is the detection range. Marked by quinaldineThe standard curve of ELISA was established for the standard, the results are shown in FIG. 4, and the relevant standard curve parameters are shown in Table 3.
TABLE 3 detection parameters of antisera (quinaldine antibodies) against quinaldine
IC 50 (μg/mL) Linear range (μg/mL) Minimum limit of detection (μg/mL) Correlation coefficient
Quinaldine antibodies 1.78502 0.43938～7.25183 0.1935 0.99073
As can be seen from FIG. 4 and Table 3, the standard curve established by taking quinaldine as a standard has a typical S-shaped curve, which shows that the detection sensitivity by using the antibody of the present invention is good, and the IC 50 1.78 μg/mL; the minimum detection limit is 0.19 mug/mL.
Example 7 specificity of antibodies
Enzyme-linked immunosorbent assay (ELISA) of quinaldine and its common fish anesthetic was performed using quinaldine antibody (Ab-hapten 2-LF, antiserum) prepared from the artificial antigen 2 (hapten 2-LF) coupled to LF of example 4, and its corresponding IC was obtained by fitting 50 And calculate the cross-reactionThe results are shown in Table 3.
TABLE 4 Cross-reactivity of antisera (quinaldine antibodies) to quinaldine and common fish anesthetics
Competitive drugs IC 50 (μg/mL) Cross reaction rate
Quinaldine 1.78502 100％
Eugenol 135.064 1.32％
Fish ballast 148.164 1.20％
Benzocaine Without any means for ＜0.01％
Procaine Without any means for ＜0.01％
As can be seen from Table 4, the cross-reactivity of quinaldine antibody to quinaldine was 100%, IC 50 The value is 1.78 mug/mL, and the eugenolThe cross reaction rate of the novel anti-tumor antibody is 1.32%, the cross reaction rate of the novel anti-tumor antibody against fish stability is 1.2%, and the cross reaction rate of the novel anti-tumor antibody against other fish anesthetics is lower than 0.01%, which shows that the novel anti-tumor antibody can specifically identify quinaldine, so that the method can detect the content of quinaldine in the fish anesthetics.
Example 8 kit for detection of quinaldine
A kit for detecting quinaldine comprises a 96-well transparent polystyrene ELISA plate coated with OVA-coupled artificial antigen 1 (hapten 1-OVA) prepared in example 2 as a coating antigen, a quinaldine standard, a quinaldine antibody (Ab-hapten 2-LF) prepared in example 4 and LF-coupled artificial antigen 2 (hapten 2-LF), horseradish peroxidase-labeled goat anti-mouse solution, a 3,3', 5' -tetramethyl benzidine (TMB) substrate color development solution, a 10% concentrated sulfuric acid stop solution, a concentrated diluent and a concentrated washing solution.
Extracting and concentrating quinaldine in a sample, adding the quinaldine sample, a quinaldine standard substance and a quinaldine antibody into a 96-hole transparent polystyrene ELISA plate which is coated with a coating raw material, incubating for 40min in a 37 ℃ water bath, washing the plate by PBST for 5 times, adding 100 mu L of a goat anti-mouse solution marked by 5000-fold dilution horseradish peroxidase, incubating for 30min in the 37 ℃ water bath, washing the plate by PBST for 5 times, adding 100 mu L of 3,3', 5' -tetramethyl benzidine (TMB) substrate chromogenic solution, incubating for 10min in the 37 ℃ water bath again, and adding 10% concentrated sulfuric acid to terminate the reaction. And quantitatively analyzing the quinaldine content in the sample by comparing the absorbance values of the sample to-be-detected liquid and the quinaldine standard.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (4)

1. A quinaldine artificial antigen is characterized by being an artificial antigen 1 or an artificial antigen 2, wherein the structural formula of the artificial antigen 1 is shown as a formula (III),
Figure FDA0004165860480000011
the structural formula of the artificial antigen 2 is shown as a formula (IV),
Figure FDA0004165860480000012
2. use of the quinaldine artificial antigen of claim 1 for the preparation of a quinaldine antibody.
3. A quinaldine artificial antigen combination comprising an artificial antigen 1 and an artificial antigen 2 of the quinaldine artificial antigen of claim 1.
4. Use of the artificial antigen combination of claim 3 for the preparation of antibodies to quinaldine and/or for the detection of quinaldine.
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