CN114591243B - Antipyrine hapten, artificial antigen, antibody and preparation methods and application thereof - Google Patents

Antipyrine hapten, artificial antigen, antibody and preparation methods and application thereof Download PDF

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CN114591243B
CN114591243B CN202210028808.5A CN202210028808A CN114591243B CN 114591243 B CN114591243 B CN 114591243B CN 202210028808 A CN202210028808 A CN 202210028808A CN 114591243 B CN114591243 B CN 114591243B
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antipyrine
ant
antibody
hapten
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CN114591243A (en
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雷红涛
方亚琳
潘康亮
全琦琪
沈兴
李向梅
王锦
关甜
徐振林
徐小艳
韦晓群
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South China Agricultural University
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Abstract

The structural formula of the antipyrine hapten is shown in a formula (I), the hapten is used for coupling carrier protein to obtain the artificial antigen, and further immune animals are used for preparing the specific antibody for detecting the antipyrine, the antibody has good specificity and detection sensitivity to the antipyrine, the semi-inhibition concentration of the antipyrine is 9.46ng/mL, the detection limit is 0.81ng/mL, the quantitative detection range is 2.01-44.48 ng/mL, and no cross reaction is caused to other structural and functional analogues, and meanwhile, the immunoassay method for detecting the antipyrine with high sensitivity, stability and rapidness is established by using the artificial antigen and the antibody, so that the method has good application background.

Description

Antipyrine hapten, artificial antigen, antibody and preparation methods and application thereof
Technical Field
The invention relates to the technical field of food detection, in particular to an antipyrine hapten, an artificial antigen, an antibody, a preparation method and application thereof.
Background
Antipyrine (ANT), also known as phenazone, is a clinical nonsteroidal antipyretic analgesic anti-inflammatory drug, belonging to pyrazolones, which mainly plays a role in antipyretic analgesic by inhibiting synthesis and release of prostaglandins. Can be used for treating fever, headache, arthralgia, neuralgia, dysmenorrhea, and active rheumatism. However, long-term or excessive use may cause abnormal blood system, such as agranulocytosis, thrombocytopenic purpura, aplastic anemia, etc., and may cause serious allergic reactions, such as severe drug eruptions, anaphylactic shock, etc., and may seriously cause gastrointestinal and acute renal failure. In recent years, illegal vendors are driven by benefits to illegally add certain western medicines with the functions of clearing heat and removing toxicity into herbal tea, wherein antipyrine is often illegally added into the herbal tea with specific cold resistance by the illegal vendors, and consumers drink the herbal tea in a large amount or for a long time without knowing, so that repeated or excessive medication can occur, and potential safety hazard to human bodies is large. The thirty-eighth plaintext rule of food safety law: the food for production and management must not be added with medicines, but substances which are traditional Chinese medicinal materials and are food can be added. The act of illegally adding antipyrine in herbal tea seriously disturbs market order and damages the health and awareness of consumers. Therefore, the establishment of the antipyrine with high sensitivity and strong specificity, which is simple and easy to be carried out, is an important technical support for guaranteeing the food safety and has important significance for guaranteeing the health of people.
At present, the detection method of antipyrine in the herbal tea published by the publication is mainly an instrument analysis method, such as high performance liquid chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry and the like. Although the methods have high accuracy and strong specificity, the pretreatment is complex, the detection cost is high, the time is long, and the methods depend on professional operators, so that the method is not beneficial to the on-site instant detection of large-scale samples. In comparison, the immune detection method has the characteristics of strong specificity, simple and convenient operation, rapidness, low cost and the like, is widely applied to detection of harmful substances in food, and can meet the supervision requirement of on-site rapid detection. The key point of the establishment of the immune detection method is to design a proper antipyrine artificial antigen and obtain an antibody with high sensitivity and strong specificity, for example, chinese patent discloses a hapten and an antigen for detecting analgin metabolite, but the detection sensitivity, the detection limit and other performances of the antibody prepared by the antigen are still to be further improved, so that the antipyrine hapten and the artificial antigen which can obtain the specific antibody with higher detection sensitivity and lower detection limit need to be developed.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide an antipyrine hapten.
The invention also aims to provide a preparation method of the antipyrine hapten.
It is a further object of the present invention to provide the use of said antipyrine hapten.
It is a further object of the present invention to provide an antipyrine artificial antigen.
It is a further object of the present invention to provide the use of said antipyrine artificial antigen.
It is a further object of the present invention to provide an antipyrine antibody.
It is still another object of the present invention to provide a kit for detecting antipyrine.
It is still another object of the present invention to provide an immunoassay for detecting antipyrine.
The above object of the present invention is achieved by the following technical solutions:
an antipyrine hapten ANT-PH, wherein the structural formula of the antipyrine hapten is shown as a formula (I):
Figure SMS_1
the hapten ANT-PH is named by a system nomenclature: 2- (4- (((1, 5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazo l-4-yl) amino) methyl) phenyl) acetic acid, i.e. 2- (4- (((1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) amino) methyl) phenyl) acetic acid.
The preparation method of the antipyrine hapten comprises the steps of reacting 4-aminoantipyrine with 2- (4- (bromomethyl) phenyl) methyl acetate in an alkaline environment, separating and purifying reactants, and hydrolyzing in the alkaline environment to obtain the antipyrine hapten shown in the formula (I).
The structural formula of the 4-aminoantipyrine is as follows:
Figure SMS_2
the structural formula of the 2- (4- (bromomethyl) phenyl) methyl acetate is as follows:
Figure SMS_3
as a preferred embodiment, the method for preparing antipyrine hapten comprises the following steps: dissolving 4-aminoantipyrine in N, N-dimethylformamide, adding anhydrous potassium carbonate to maintain an alkaline environment for the whole reaction system, adding 2- (4- (bromomethyl) phenyl) methyl acetate, heating to 50 ℃, condensing and refluxing for reaction for 4 hours; and (3) spin-drying the reaction product obtained after separation and purification, separating and purifying the reactant, dissolving the separated and purified reactant in methanol, stirring the methanol and a sodium hydroxide aqueous solution for 3-5 h at room temperature, and adjusting the pH value to 6-7 after the reaction is finished to obtain the hapten ANT-PH.
Preferably, the molar ratio of the 4-aminoantipyrine to the methyl 2- (4- (bromomethyl) phenyl) acetate is 1.1 to 2.
Further preferably, the molar ratio of 4-aminoantipyrine to methyl 2- (4- (bromomethyl) phenyl) acetate is 1:1.2.
Preferably, the molar ratio of the 4-aminoantipyrine to the potassium carbonate is 1-1.5:4-6.
Further preferably, the molar ratio of 4-aminoantipyrine to potassium carbonate is 1:4.
Preferably, the concentration of the sodium hydroxide aqueous solution is 1mol/L.
The invention also provides application of the antipyrine hapten in preparation of antipyrine artificial antigen.
An antipyrine artificial antigen, the structural formula of which is shown as a formula (II):
Figure SMS_4
wherein protein is a carrier protein.
Preferably, the carrier protein is selected from bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (LF) or chicken Ovalbumin (OVA).
The preparation method of the antipyrine artificial antigen is that the antipyrine hapten carboxyl coupling carrier protein shown in the formula (I) is obtained.
Preferably, the coupling is by the active ester method.
As a preferred embodiment, the method for preparing the antipyrine artificial antigen comprises the following steps:
(1) Dissolving ANT-PH and N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring at room temperature for 2-4 hours in a dark place to obtain an ANT-PH activating solution;
(2) The carrier protein was added to PBS buffer (0.01 mol/L, pH=7.4);
(3) Slowly and dropwise adding the ANT-PH activating solution in the step (1) into the carrier protein buffer solution in the step (2), and reacting for 12 hours at 4 ℃;
(4) And (3) dialyzing the reaction solution obtained in the step (3) by using PBS buffer solution to obtain the antipyrine artificial antigen.
Preferably, in the step (1), the mass ratio of the ANT-PH, the NHS and the EDC is 1-2:1-2:2-3.
More preferably, the mass ratio of ANT-PH, NHS to EDC in step (1) is 1.3:1.6:2.5.
Preferably, the ratio of the carrier protein to PBS buffer in step (2) is 5 mg/2 mL by mass.
Preferably, the mass ratio of ANT-PH in step (1) to the carrier protein in step (2) is 1-5:3-8.
More preferably, the mass ratio of ANT-PH in step (1) to the carrier protein in step (2) is 1:4.
The invention also provides application of the antipyrine artificial antigen in preparation of antipyrine antibodies.
An antipyrine antibody is prepared by immunizing an animal with any antipyrine artificial antigen.
Preferably, the antipyrine antibody is prepared by immunizing an animal with antipyrine artificial antigen (ANT-PH-BSA) using a carrier protein as Bovine Serum Albumin (BSA).
Further preferably, the antipyrine antibody is a monoclonal antibody or a polyclonal antibody.
As a preferred embodiment, the method for preparing an antipyrine polyclonal antibody comprises the steps of:
(1) The prepared artificial antigen of ANT-PH coupled bovine serum albumin (ANT-PH-BSA) is used as immunogen to be uniformly emulsified with an equal amount of immunological adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the later booster immunization), so as to immunize animals. 2.5 kg to 3kg of New Zealand white rabbits are immunized by adopting various injection modes of subcutaneous back, subcutaneous leg muscle and ear vein, and the immunization is carried out for the second time after 4 weeks, and then the immunization is enhanced once every 3 weeks. The ear margin vein was bled 1 week after the third boost and serum titers were determined using an indirect competition ELISA. When the potency no longer increases, the ear margin vein is used to boost the immunity.
(2) After one week of booster immunization, the heart takes blood, water bath is carried out for 0.5 to 1h, centrifugation is carried out for 15min at 4 ℃ and 10000rpm/min, and the supernatant is taken as antiserum. The antiserum was purified by ammonium sulfate precipitation to polyclonal antibodies.
The antipyrine polyclonal antibody prepared by the method is also within the protection scope of the invention.
The application of the antipyrine antibody in detecting antipyrine and/or preparing a reagent kit for detecting antipyrine is also within the protection scope of the invention.
A kit for detecting antipyrine comprises the antipyrine artificial antigen and an antibody prepared by immunizing an animal with the antipyrine artificial antigen.
Preferably, the kit comprises an antipyrine artificial antigen (ANT-PH-OVA) taking carrier protein as chicken Ovalbumin (OVA) and an antipyrine artificial antigen (ANT-PH-BSA) taking carrier protein as Bovine Serum Albumin (BSA) for immunizing an animal.
Preferably, the kit further comprises one or more of an enzyme label plate, an antipyrine standard, an enzyme conjugate, a chromogenic solution, a stop solution or a washing solution.
Further preferably, the kit further comprises an enzyme label plate coated with the antipyrine artificial antigen, an antipyrine standard solution, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color development solution, a stop solution and a washing solution.
Further preferably, the enzyme conjugate is a horseradish peroxidase-labeled antipyrine antibody.
Still more preferably, the antibody is a polyclonal antibody obtained by immunizing an animal with an antipyrine artificial antigen (ANT-PH-BSA) in which the carrier protein is Bovine Serum Albumin (BSA).
Further preferably, the kit adopts an indirect competition ELISA method, the enzyme label plate microporous strip is pre-coated with the coating antigen, the antipyrine remained in the sample and the pre-coated enzyme conjugate of the antipyrine of the coating antigen on the enzyme label plate microporous strip are subjected to color development by using a TMB substrate, the absorbance value of the sample is in negative correlation with the content of the residual antipyrine contained in the sample, and the sample is compared with a standard curve and multiplied by the corresponding dilution factor to obtain the residual antipyrine in the sample.
Preferably, the antipyrine standard solution has 8 concentration gradients of 1000 μg/L,200 μg/L,40 μg/L,8 μg/L,1.6 μg/L,0.32 μg/L,0.064 μg/L, and 0.0128 μg/L, respectively.
Preferably, the color development liquid consists of substrate liquid A and substrate liquid B, wherein the liquid A is hydrogen peroxide or carbamide peroxide, and the liquid B is o-phenylenediamine or tetramethyl benzidine.
Preferably, the stop solution is a sulfuric acid solution of 1-2 mol/L.
Preferably, the washing liquid is phosphate buffer solution with the pH value of 7.4 and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L; the percentages are by weight volume.
Preferably, the preparation method of the ELISA plate comprises the following steps: diluting the coating raw material into 50 mug/L by using a coating buffer solution, adding 100 mug/L into each hole, incubating overnight at 37 ℃ in a dark place, pouring out liquid in the holes, washing 2 times by using a washing solution for 30s each time, beating to dryness, adding 150-200 mug of sealing solution into each hole, incubating for 1-2 h at 25 ℃ in a dark place, pouring out liquid in the holes, beating to dryness, drying and vacuum sealing by using an aluminum film for storage.
Preferably, the coating buffer solution used in the preparation process of the ELISA plate is carbonate buffer solution with the pH value of 9.6 and 0.05 mol/L; the blocking solution is phosphate buffer solution with the pH value of 7.1-7.5 and contains 1-3% casein and 0.1-0.3 mol/L; the percentages are by weight volume.
An immunoassay method for detecting antipyrine, which uses the antipyrine artificial antigen as an antigen and uses the antipyrine antibody as a detection antibody for detection; the immunoassay method is a method of non-diagnostic therapeutic interest.
Preferably, the detection is carried out by taking an antipyrine artificial antigen (ANT-PH-OVA) with a carrier protein as chicken Ovalbumin (OVA) as a coating antigen, and taking an antipyrine artificial antigen (ANT-PH-BSA) with a carrier protein as Bovine Serum Albumin (BSA) as an immunogen to immunize an animal to obtain an antibody as a detection antibody.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
An antipyrine colloidal gold rapid detection test strip comprises a base plate, and a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged on the base plate, wherein an invisible detection line and an invisible quality control line are printed on the nitrocellulose membrane, the invisible detection line is printed by adopting a coating antigen solution, and the invisible quality control line is printed by adopting a goat anti-rabbit antibody; the coating antigen is any antipyrine artificial antigen.
Preferably, the coating antigen is ANT-PH-OVA.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a new antipyrine hapten, an antipyrine artificial antigen is prepared by using the hapten, and a specific antibody for detecting antipyrine is prepared by further immunizing animals, wherein the antibody has good specificity and detection sensitivity to antipyrine, the detection limit to the antipyrine half-inhibition concentration is 9.46ng/mL, the detection limit is 0.81ng/mL, the quantitative detection range is 2.01-44.48 ng/mL, and no cross reaction is caused to analgin, phenylbutazone, aspirin, ibuprofen and other structural and functional analogues, so that a core raw material is provided for establishing an immune detection method of the specific antipyrine; an immunoassay method of antipyrine with higher specificity and sensitivity is established; in addition, the polyclonal antibody is utilized to develop a kit for detecting the antipyrine residues, and the kit has the characteristics of high specificity, high sensitivity, high accuracy and the like.
Drawings
FIG. 1 is a synthetic scheme for the antipyrine hapten (ANT-PH) of example 1 of the present invention.
FIG. 2 is an ultraviolet scan of ANT-PH, BSA, ANT-PH-BSA of example 2 of the present application.
FIG. 3 is an ANT-PH, OVA, ANT-PH-OVA ultraviolet scan of example 2 of the present application.
FIG. 4 is an ANT-PH, LF, ANT-PH-LF ultraviolet scan of example 2 of the present application.
FIG. 5 is an indirect competition ELISA standard curve for antipyrine antibodies of example 5 of the present application.
FIG. 6 is a schematic structural diagram of an antipyrine colloidal gold rapid detection test strip according to embodiment 8 of the present application; wherein: 1. a PVC plastic bottom plate; 2. a sample pad; 3. nitrocellulose membrane (NC membrane); 4. a water absorbing pad; 5. a test line; 6. and a control line.
FIG. 7 is a graph showing the determination of the result of the rapid detection test strip of antipyrine colloidal gold in example 8 of the present application; wherein: a is a negative sample detection result, B is a positive sample detection result, and C and D are test strip failures.
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 preparation and identification of an antipyrine hapten
1. Preparation of antipyrine hapten ANT-PH
203mg (1 mmol) of 4-aminoantipyrine was charged into a 50mL round bottom flask, 3mL of N, N-dimethylformamide was added for dissolution, 553mg (4 mmol) of anhydrous potassium carbonate was then added to maintain an alkaline environment for the whole reaction system, and 206. Mu.L (1.2 mmol) of methyl 2- (4- (bromomethyl) phenyl) acetate was further added and heated to 50℃for condensation reflux reaction for 4 hours. The reaction was monitored by TLC until completion, and the developing solvent was petroleum ether-ethyl acetate (3:1, v/v). Purifying the obtained reaction product by silica gel column chromatography, extracting with ultrapure water and ethyl acetate at a ratio of 1:1, concentrating the ester layer by a rotary evaporation evaporator, and obtaining the antipyrine hapten (ANT-PH). The synthetic scheme of ANT-PH is shown in FIG. 1.
2. Identification of antipyrine hapten ANT-PH
Nuclear magnetic resonance hydrogen spectrum results of ANT-PH: 1 H NMR(600MHz,Methanol-d4)δ 9.57(d,1H),7.43-7.37(m,4H),7.33-7.28(m,4H),7.27-7.22(m,1H), 4.40(d,2H),4.00(q,J＝7.1Hz,3H),3.21(p,J＝1.7Hz,3H),2.11(s,3H)。
the mass spectrum identification result of ANT-PH is: MS: c (C) 20 H 21 N 3 O 3 ：351.16，ESI+[M-H]+:352.3。
According to the nuclear magnetic resonance hydrogen spectrum and the mass spectrum result, the derivation site is correct and successful, which shows that the antipyrine hapten ANT-PH of the target product is successfully formed, and the structural formula is shown as the formula (I):
Figure SMS_5
hapten ANT-PH was named by systematic nomenclature: 2- (4- (((1, 5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazo l-4-yl) amino) methyl) phenyl) acetic acid, i.e. 2- (4- (((1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) amino) methyl) phenyl) acetic acid.
Example 2 preparation and identification of an antipyrine Artificial antigen
1. Preparation of antipyrine artificial antigen
Different artificial antigens were prepared by coupling the antipyrine hapten (ANT-PH) prepared in example 1 to Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA) and Lactoferrin (LF) by the active ester method, respectively.
(1) Respectively weighing 10mg of ANT-PH prepared in example 1, 2mg of NHS and 3mg of EDC, dissolving in 50-100 mu L of DMF, and stirring at room temperature in a dark place for 2-4 hours to obtain antipyrine hapten activating solution;
(2) 15mg of Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA) and Lactoferrin (LF) were weighed separately and added to 1mL of PBS buffer (0.01 mol/L, pH=7.4);
(3) Slowly adding the antipyrine hapten activating solution obtained in the step (1) into the carrier protein buffer solution obtained in the step (2) dropwise, and stirring for 12 hours at 4 ℃;
(4) Dialyzing with PBS buffer solution for two days and 4 times per day, and obtaining antipyrine artificial antigen (ANT-PH-BSA, ANT-PH-OVA, ANT-PH-LF) after dialyzing, packaging in centrifuge tube, and preserving at-20deg.C for use.
Wherein, the formula of Phosphate Buffered Saline (PBS): na (Na) 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.50g， KCl 0.20g，KH 2 PO 4 0.20g, distilled water was added to a volume of 1000mL.
2. Identification of antipyrine artificial antigen
The above synthesized antipyrine artificial antigens (ANT-PH-BSA, ANT-PH-OVA, ANT-PH-LF) were subjected to ultraviolet full-wave scanning, and the results are shown in FIG. 2, FIG. 3, and FIG. 4.
Specifically, BSA, ANT-PH and ANT-PH-BSA were identified by ultraviolet (200-400 nm) scanning, respectively, and the absorption curve of the antipyrine immunogen ANT-PH-BSA was found to be significantly different from that of the carrier protein BSA by comparing the highest absorbance values of the substances before and after coupling, the ANT-PH had a characteristic peak at 330nm, and after coupling reaction, the absorption peaks of the ANT-PH-BSA were significantly higher than that of BSA at 230nm and 280nm, and a significant shift was seen by comparing the curves of the ANT-PH (FIG. 2). Since the small molecule components such as unreacted drugs are removed by dialysis after the coupling, the drug characteristic peak of the coupled product is contributed by the protein-bound drug molecules, thus indicating that the reaction product is a complex of carrier protein BSA and ANT-PH, and the coupling of ANT-PH-BSA is successful.
Specifically, OVA, ANT-PH and ANT-PH-OVA were respectively identified by ultraviolet (200-400 nm) scanning, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the antipyrine artificial antigen ANT-PH-OVA was significantly different from that of the carrier protein OVA, the ANT-PH had a characteristic peak at 330nm, and after the coupling reaction, the absorption peaks of the ANT-PH-OVA were significantly higher than that of OVA at 230nm and 280nm, and a significant shift was seen by comparing the curves of the ANT-PH (FIG. 3). Since the small molecule components such as unreacted drugs are removed by dialysis after the coupling, the drug characteristic peak of the coupled product is contributed by the protein-bound drug molecules, thus indicating that the reaction product is a complex of carrier protein OVA and ANT-PH, and the ANT-PH-OVA coupling is successful.
Specifically, LF, ANT-PH and ANT-PH-LF were respectively identified by ultraviolet (200-400 nm) scanning, and the absorption curve of the artificial antigen ANT-PH-LF of antipyrine was found to be significantly different from that of the carrier protein LF by comparing the highest absorbance values of the substances before and after coupling, the ANT-PH had a characteristic peak at 330nm, and after coupling reaction, the absorption peaks of ANT-PH-LF were significantly higher than LF at 230nm and 280nm, and a significant shift was seen by comparing the curves of ANT-PH (FIG. 4). Since the small molecular components such as unreacted drugs are completely removed by dialysis after the coupling, the drug characteristic peak of the coupled product is contributed by the protein-bound drug molecules, so that the reaction product is a complex of carrier proteins LF and ANT-PH, and the ANT-PH-LF coupling is successful.
Example 3 preparation of antibodies
1. Preparation of polyclonal antibodies
Antipyrine artificial antigens ANT-PH-BSA and ANT-PH-coupled Lactoferrin (LF) prepared in example 2 were used as immunogens respectively, and were uniformly emulsified with an equivalent amount of an immunoadjuvant (Freund's complete adjuvant for the first immunization, freund's incomplete adjuvant for the subsequent booster immunization), respectively, to immunize animals. 2.5 kg to 3kg of New Zealand white rabbits are immunized by adopting various injection modes of subcutaneous back, subcutaneous leg muscle and ear vein, and the immunization is carried out for the second time after 4 weeks, and then the immunization is enhanced once every 3 weeks. The ear margin vein was bled 1 week after the third boost and serum titers were determined using an indirect competition ELISA. When the potency no longer increases, the ear margin vein is used to boost the immunity. After one week of booster immunization, the heart takes blood, water bath is carried out for 0.5 to 1h, centrifugation is carried out for 15min at 4 ℃ and 10000rpm/min, and the supernatant is taken as antiserum. The antiserum is purified by adopting an ammonium sulfate precipitation method to obtain a polyclonal antibody, and the polyclonal antibody is frozen at the temperature of minus 20 ℃ for standby.
2. Preparation of monoclonal antibodies
Antipyrine artificial antigens ANT-PH-BSA and ANT-PH-coupled lactoferrin LF prepared in example 2 are respectively used as immunogens, and are uniformly emulsified with an equal amount of immune adjuvant (complete Freund's adjuvant is used for the first immunization and incomplete Freund's adjuvant is used for the later boosting), so that a mouse is immunized by adopting an abdominal subcutaneous multipoint injection method, the serum titer is detected by adopting tail venous blood of the mouse after 1 week of boosting, the boosting is carried out again after the antibody titer is no longer increased, and the spleen cells of the mouse are fused with myeloma cells of the mouse after 7 days. The complete medium is used for cell culture after the hybridoma cells are screened out from the HAT medium. Detecting cell supernatant by using an ic-ELISA method, cloning and culturing the cells in the holes with strong positive detection results by using a limiting dilution method, detecting again after one week, picking the holes and recloning. After 3 times of clone culture detection, the hybridoma cell of the monoclonal antibody is obtained. After the hybridoma cells are cultured in an enlarged manner, the hybridoma cells are inoculated into the abdominal cavity of a mouse, and ascites containing the antibody is produced. The ascites is purified by caprylic acid-ammonium sulfate precipitation method to obtain monoclonal antibody, and frozen at-20deg.C for use.
Example 4 screening of antipyrine immunogens and coating precursors
Screening of coating antigen by indirect competition ELISA method using the antibodies obtained after immunization of animals with ANT-PH-BSA and ANT-PH-LF of example 3, respectively, and artificial antigens ANT-PH-BSA, ANT-PH-LF and ANT-PH-OVA prepared in example 2 as coating antigen; the optimal immunogen and coating antigen were selected by indirect competition ELISA method to obtain serum titers and inhibition rates.
An indirect competition ELISA method for screening antipyrine immunogens and coating immunogens comprising the steps of:
(1) The artificial antigens ANT-PH-BSA, ANT-PH-OVA and ANT-PH-LF prepared in example 2 were used as coating sources, diluted to 1. Mu.g/mL with coating solution, coated on 96-well ELISA plates, and incubated overnight (12 h) at 37℃with 100. Mu.L of each well;
(2) Removing the coating liquid, washing for 2 times, and beating to dry;
(3) 120 mu L of sealing liquid (namely 1% of fish skin collagen) is added into each hole, and the mixture is sealed for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, and taking out;
(5) The antipyrine polyclonal antibodies prepared in example 3 were diluted in a multiple ratio to seven gradients with PBST, i.e. 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, while blank control wells (replaced with PBST) were set; diluting the antipyrine drug to 1 mug/mL;
(6) The titers are as follows: adding 50 mu L of PBST into each hole, sequentially adding 50 mu L of the obtained antibody into the holes according to the ratio of the PBST to each hole, and replacing the last hole with 50 mu L of PBST without adding the antibody;
inhibition column: adding 50 mu L of medicine into each hole, diluting the obtained antibody by multiplying ratio, sequentially adding 50 mu L of the obtained antibody into the holes, adding no antibody into the last hole, and replacing the last hole with 50 mu L of PBST; incubating at 37deg.C for 40min, washing for 5 times, and drying;
(7) Adding goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μl/well, incubating at 37deg.C for 30min, washing five times, and drying;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 2mol/L H are added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
The potency is OD 450 The dilution factor of the antibody is about 1.0.
Inhibition = (OD value of potency-OD value of inhibition)/OD value of inhibition × 100%.
The serum titers and inhibition results for the different immunogen and coating combinations are shown in table 1.
TABLE 1 serum titers and inhibition rates of different immunogens in combination with coating antigen
Figure SMS_6
As can be seen from table 1, antibodies produced by new zealand white rabbits immunized with different antipyrine artificial antigens as immunogens all have certain titers; meanwhile, the obtained antibody has different degrees of inhibition effects on the target analyte antipyrine. The potency of the combination of the immunogen and the coating antigen of the number 1 is 1:256000 and the inhibition rate is 89.87 percent, which are higher than those of the combination of the immunogen and the coating antigen of the numbers 2,3 and 4, so that the immunogen and the coating antigen of the number 1 can not only specifically identify the target analyte antipyrine, but also have good antibody sensitivity, so that ANT-PH-BSA is used as the optimal immunogen, and ANT-PH-OVA is used as the optimal coating antigen.
Example 5 establishment of an Indirect competitive ELISA detection method for antipyrine
1. Experimental method
An indirect competition ELISA method to detect antipyrine comprising the steps of:
(1) The artificial antigen ANT-PH-OVA prepared in example 2 was used as a coating source, diluted to 50. Mu.g/L with a coating solution, and a 96-well ELISA plate was coated, 100. Mu.L of the coating solution was added to each well, and incubated overnight (12 h) at 37 ℃;
(2) Removing the coating liquid, washing for 2 times, and beating to dry;
(3) 120 mu L of sealing liquid (namely 1% of fish skin collagen) is added into each hole, and the mixture is sealed for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, and taking out;
(5) The antipyrine polyclonal antibody prepared in example 3 was diluted 16000-fold with PBST, and antipyrine standard was diluted to 1000 μg/L,200 μg/L,40 μg/L,8 μg/L,1.6 μg/L,0.32 μg/L,0.064 μg/L,0.0128 μg/L;
(6) 50 mu L of antipyrine drug diluent (three groups are parallel) is added into each row, 50 mu L of antipyrine polyclonal antibody diluent prepared in the example 3 is added into each row, incubation is carried out for 40min at 37 ℃, washing is carried out for 5 times, and beating is carried out;
(7) Adding goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μl/well, incubating at 37deg.C for 30min, washing five times, and drying;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 2mol/L H are added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
2. Experimental results
An indirect competition ELISA standard curve of the antibodies for detecting the antipyrine is shown in FIG. 5, and the semi-inhibition concentration IC of the antipyrine polyclonal antibody prepared in example 3 to the antipyrine can be known from FIG. 5 50 9.46/ng/mL, quantitative detection of linear Range IC 20 ～IC 80 2.01-44.48 ng/mL, and the detection limit is 0.81ng/mL; the invention proves that the antibody for detecting the antipyrine prepared by the invention can meet the detection requirement and has high recognition capability on the antipyrine.
Example 6 evaluation of specificity of antibodies for detection of antipyrine
1. Experimental method
The cross-reactivity of the polyclonal antibody prepared in example 3 to other structural and functional analogues was determined by replacing the drug antipyrine in example 5 with analgin, phenylbutazone, aspirin and ibuprofen and performing the above-described test at the same dilution.
The specificity of the antibodies used for detecting the antipyrine is determined by carrying out a cross-reaction experiment on the antipyrine and structural and functional analogues thereof, wherein the specificity of the antibodies is expressed by a cross-reaction rate (CR), and the smaller the cross-reaction rate is, the stronger the specificity is. The antipyrine structure and its functional analogues (analgin, phenylbutazone, aspirin and ibuprofen) were diluted by multiple ratios, and measured by indirect competition ELISA, and the IC of each structural and functional analogue was obtained by the same sensitivity verification method as in example 5 50 Values, antipyrine cross-reactivity (CR) was calculated according to the following formula:
CR(％)＝IC 50 (antipyrine)/IC 50 (structural and functional analogues) ×100%
2. Experimental results
The results of cross-reactions of antipyrine with its structural and functional analogues are shown in table 2.
TABLE 2 Cross-reaction results of antipyrine and its structural and functional analogues
Figure SMS_7
Note that: NR indicates no reaction.
As can be seen from table 2, the antibodies used to detect antipyrine did not cross react with analgin, phenylbutazone, aspirin and ibuprofen; the polyclonal antibody prepared in the example 3 for detecting the antipyrine has strong specificity to the antipyrine, can effectively eliminate the interference of structural and functional analogues (analgin, phenylbutazone, aspirin and ibuprofen) to the antipyrine, and can be specially used for detecting the antipyrine.
EXAMPLE 7 development of ELISA kits for the detection of antipyrine
1. A kit for detecting antipyrine is constructed, the kit comprising the following parts:
(1) Preparing an ELISA plate coated with a coating source: the artificial antigen ANT-PH-OVA prepared in example 2 is used as a coating source, the coating source is diluted to 50 mug/L by using a coating buffer solution, 100 mug/L is added into each hole, the mixture is incubated overnight at 37 ℃ in a dark place, liquid in the hole is poured out, the mixture is washed for 2 times by using a washing solution for 30 seconds each time, the mixture is patted dry, then 200 mug of sealing solution is added into each hole, the mixture is incubated for 2 hours at 25 ℃ in a dark place, the liquid in the hole is poured out, the mixture is patted dry, and the mixture is vacuum-sealed and stored by using an aluminum film; the coating buffer solution is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, the sealing solution is phosphate buffer solution with the pH value of 7.1-7.5 and contains 1-3% casein and 0.1-0.3 mol/L;
(2) Antipyrine standard solution: 8 concentration gradients of 1000 μg/L,200 μg/L,40 μg/L,8 μg/L,1.6 μg/L,0.32 μg/L,0.064 μg/L,0.0128 μg/L, respectively;
(3) An antipyrine polyclonal antibody prepared in example 3;
(4) Enzyme conjugate: an antipyrine polyclonal antibody prepared in example 3 labeled with horseradish peroxidase;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) H with termination liquid of 2mol/L 2 SO 4 ；
(7) The washing liquid is pH 7.4, contains 0.5-1.0% Tween-20, 0.01-0.03% sodium azide preservative and 0.1-0.3 mol/L phosphate buffer solution, and the percentages are weight volume percentages.
2. Actual sample detection
And numbering corresponding micropores of the sample and the standard substance in sequence, making 2 holes of each sample and each standard substance in parallel, and recording the positions of the standard holes and the sample holes. The enzyme conjugate concentrate is diluted with an enzyme conjugate diluent in a 1:10 volume ratio (i.e., one portion of enzyme conjugate concentrate is added to 10 portions of enzyme conjugate diluent, ready for use) as desired. Adding 50 mu L of standard substance/sample into corresponding microwells, adding 50 mu L of enzyme conjugate working solution, gently shaking and mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 30min after covering with a cover plate film. The liquid in the well was dried and 250. Mu.L/well of the washing liquid was added. Washing for 4-5 times, and at intervals of 10s each time, pouring out the washing liquid in the plate holes, and drying by using absorbent paper (after drying, the unused gun heads can be used for puncturing by the bubbles which are not clear). Adding substrate color development solution A50 mu L/hole, adding substrate color development solution B50 mu L/hole, mixing with gentle shaking, placing the mixture in a light-shielding environment at 25deg.C for reaction for 10min, adding stop solution 50 mu L/hole, mixing with gentle shaking, setting enzyme-labeling instrument and 450nm, and measuring OD value of each hole.
3. Analysis of detection results
The percent absorbance of a standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard) and multiplied by 100%. And drawing a standard curve graph by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mug/L) of the antipyrine standard substance as an abscissa. Substituting the percentage absorbance of the sample into a standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the dilution factor corresponding to the standard curve to obtain the actual concentration of the antipyrine in the sample.
Example 8 development of an antipyrine colloidal gold rapid test strip
1. Assembly of colloidal gold rapid detection test strip
As shown in fig. 6, the colloidal gold rapid test strip is formed by superposing a nitrocellulose membrane (NC membrane) 3, a sample pad 2, a water absorbing pad 4 and a PVC plastic base plate 1. Specifically, a coating antigen (ANT-PH-OVA) and goat anti-rabbit IgG were sprayed on an NC film with a spray amount of 0.8. Mu.L/cm using an XYZ three-dimensional spray point film cutter as a test line 5 and a control line 6 (T line and C line) which were located in the middle of the NC film 3 and spaced apart from each other by 6mm, dried at 37℃for 12 hours, and then the cellulose film 3 was stuck on the middle portion of a backing plate, the sample pad 2 overlapped with the T line 5 end of the NC film by 1mm, and a water absorbing pad 4 was stuck on the upper side of the cellulose film 3 overlapped with the cellulose film 3 by 1mm, and the assembled test paper board was cut into test strips 3.05mm wide using a chopper.
2. Preparation of gold-labeled antibody
Colloid Jin Xuanfu with average diameter of 30nm is prepared by reducing chloroauric acid with trisodium citrate. 1mL of colloidal gold solution is taken and added with 0.2mol/L K 2 CO 3 The pH of the solution was adjusted to about 8.0, 10. Mu.g of antibody was added for incubation for 30min, and after incubation for 30min with 10% BSA solution, centrifugation was performed at 10000rpm for 20min at 4℃and the supernatant was removed, resuspended with 200. Mu.L of 0.2mol/L phosphate buffer pH 7.4 (containing 0.5% Tween-20, 0.5% BSA, 5% sucrose, 0.3% PVP, 0.03% procline-300) and stored at 4 ℃.
3. Detection of herbal tea samples
(1) Herbal tea sample extraction and purification
Sucking 0.5mL of herbal tea, adding 4.5mL of 0.2mol/L phosphate buffer solution with pH of 7.4, diluting, and vortex mixing for 30s to obtain the liquid to be tested.
(2) Detection step
And (3) adding 150 mu L of sample to-be-detected liquid into the micropores, adding 5 mu L of gold-labeled antibody, repeatedly sucking and beating, uniformly mixing, incubating for 5min at room temperature, inserting a test strip into the micropores, reacting for 3min, taking out the test strip, removing a sample pad, and judging the result.
(3) Determination of detection results
As shown in FIG. 7, if the sample does not contain antipyrine as an analyte, the gold-labeled antibody is combined with the coating antigen on the test line (T line) on the strip for rapid detection, so that the test line shows a clear red line, namely the detected sample is negative (as shown in FIG. 7A); if the sample contains the antipyrine to be detected, the antipyrine is combined with the gold-labeled antibody and cannot be captured by a test line on the rapid detection test strip, and the test line is positive when not developed (as shown in FIG. 7B); similarly, the gold-labeled antibody is also combined with goat anti-rabbit IgG on a quality control line (C line) on the cellulose membrane, so that the quality control line is red, and the presence or absence of the color of the quality control line indicates that the test strip is effective or ineffective (as shown in FIG. 7A, B, effective C, D is ineffective). And judging the detection result within 3-5 minutes.
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 (3)

1. The antipyrine artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling antipyrine hapten with bovine serum albumin, and the coating antigen is obtained by coupling antipyrine hapten with chicken egg albumin; the structural formula of the antipyrine hapten is shown as a formula (I):
Figure QLYQS_1
formula (I).
2. The antipyrine artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling antipyrine hapten with bovine serum albumin, and the coating antigen is obtained by coupling antipyrine hapten with lactoferrin; the structural formula of the antipyrine hapten is shown as a formula (I):
Figure QLYQS_2
formula (I).
3. The antipyrine artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling antipyrine hapten with lactoferrin, and the coating antigen is obtained by coupling antipyrine hapten with chicken egg albumin; the structural formula of the antipyrine hapten is shown as a formula (I):
Figure QLYQS_3
formula (I).
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