CN114605322B - Dextromethorphan hapten, artificial antigen and antibody as well as preparation methods and application thereof - Google Patents

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

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CN114605322B
CN114605322B CN202210182915.3A CN202210182915A CN114605322B CN 114605322 B CN114605322 B CN 114605322B CN 202210182915 A CN202210182915 A CN 202210182915A CN 114605322 B CN114605322 B CN 114605322B
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dxm
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雷红涛
方亚琳
全琦琪
潘康亮
王子安
关甜
王锦
沈兴
李向梅
韦晓群
徐小艳
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Abstract

The invention provides a dextromethorphan hapten, an artificial antigen, an antibody and a preparation method and application thereof, wherein the structural formula of the dextromethorphan hapten is shown as a formula (I), the dextromethorphan hapten is used for coupling carrier protein to obtain the artificial antigen, and further an immune animal is used for preparing a specific antibody for detecting dextromethorphan, the artificial antigen is used as a coating source, the antibody has good specificity and detection sensitivity to dextromethorphan, the half-inhibition concentration of the antibody to dextromethorphan is 4.42ng/mL, the detection limit is 0.18ng/mL, the quantitative detection range is 0.59-33.37 ng/mL, and no cross reaction is caused to other structural analogues and functional analogues, and meanwhile, the antibody is used for establishing an immunoassay method for detecting dextromethorphan with high sensitivity, stability and rapidness, so that the rapid detection of dextromethorphan is realized.

Description

Dextromethorphan hapten, artificial antigen and antibody as well as preparation methods and application thereof
Technical Field
The invention relates to the technical field of food detection, in particular to dextromethorphan hapten, artificial antigen and antibody as well as a preparation method and application thereof.
Background
Dextromethorphan (DXM), also known as Dextromethorphan, is the dextrorotatory isomer of morphine-like levorphanol methyl ether. Clinically, the over-the-counter cough suppressant is an efficient and widely used over-the-counter cough suppressant, and has an effect of suppressing the center of cough with bulbar, and is mainly used for treating common cold, sphagitis, acute or chronic bronchitis, cough caused by upper respiratory tract infection and the like. Tolerance and addiction are not seen at the therapeutic dose, and the effect is fast and safe. Despite the significant advantages in clinical treatment, dextromethorphan dependence and abuse in the human population gradually appear as dextromethorphan is widely used. The excessive dose of dextromethorphan can cause euphoria, illusion, excitation impulse and dissociative sedation of human body, and withdrawal symptoms such as dizziness, anxiety, insomnia, cognitive mood disorder and the like appear after the administration, thus showing obvious drug dependence.
Recently, it has been reported that, in order to improve the efficacy of herbal tea to obtain greater commercial profits, lawsons illegally incorporate dextromethorphan into herbal tea purported to have potent antitussive effects. The illegally added medicine of the on-site herbal tea is mixed at random and various dosage, and the incompatibility is not noticed, and the special pharmacist does not instruct the use and the dosage limitation during the sales. The consumers drink in a large quantity or for a long time without awareness, repeated or overdose situations can occur, and the potential safety hazard is high. This illegal activity severely violates food safety laws and brings potential health threats to consumers without awareness.
At present, the detection method for dextromethorphan mainly comprises an instrument analysis method such as high performance liquid chromatography, capillary gas chromatography, liquid chromatography-mass spectrometry and the like, has the advantages of high accuracy, high sensitivity and the like, but the instrument and the equipment are expensive, require professional operators to operate, cannot meet the requirement of on-site rapid detection, and are difficult to popularize in a base layer. Therefore, the detection method of dextromethorphan in the herbal tea with simplicity, easiness, high travelling sensitivity and strong specificity is established, is an important technical support for guaranteeing food safety, and has important significance for guaranteeing physical health of people. The immune method has low cost and simple and convenient operation, plays an irreplaceable role in the field of on-site rapid screening, and has the key technology of obtaining the antibody with high sensitivity and specificity. However, currently there is a lack of highly sensitive and specific dextromethorphan antibodies, dextromethorphan hapten and artificial antigen useful for producing highly sensitive and specific antibodies.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of a dextromethorphan detection method in the prior art and provides dextromethorphan hapten, artificial antigen and antibody as well as preparation methods and application thereof.
The invention aims to provide dextromethorphan hapten.
The invention also aims to provide a preparation method of the dextromethorphan hapten.
The invention also aims to provide application of the dextromethorphan hapten in preparation of dextromethorphan artificial antigens.
The invention also aims to provide the dextromethorphan artificial antigen.
The invention also aims at providing a preparation method of the dextromethorphan artificial antigen.
The invention also aims to provide application of the dextromethorphan artificial antigen in preparation of dextromethorphan artificial antibodies.
The invention also aims to provide a dextromethorphan antibody.
The invention also aims to provide a kit for detecting dextromethorphan.
The invention also aims to provide an immunoassay method for detecting dextromethorphan.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a hapten DXM-2C of dextromethorphan, wherein the structural formula of the hapten DXM-2C is shown as a formula (I):
Figure BDA0003521971410000021
the hapten DXM-2C is named as follows by adopting a system naming method: 2- (((4 bS,8aS, 9S) -11-methyl-6,7, 8a,9,10-hexahydro-5H-9,4b- (epiminoethane-3-yl) oxy) acetic acid, i.e., 2- (((4 bS,8aS, 9S) -11-methyl-6,7, 8a,9,10-hexahydro-5H-9,4b- (iminoethanol) phenanthran-m-3-yl) oxy) acetic acid.
The hapten DXM-2C is prepared by reacting dextromorphic acid with ethyl bromoacetate in a solvent, separating and purifying reactants, and hydrolyzing in an alkaline environment.
The structural formula of the dextromorphan is as follows:
Figure BDA0003521971410000031
the structural formula of the ethyl bromoacetate is as follows:
Figure BDA0003521971410000032
specifically, dextromorphan, potassium carbonate and ethyl bromoacetate are dissolved in a solvent, stirred, condensed and refluxed at 40 ℃ for 4-8 hours, separated and purified reactants are dissolved in methanol, then sodium hydroxide aqueous solution is added, stirred for 4-6 hours at room temperature, and hydrochloric acid solution is used for regulating acid after the reaction is finished, thus obtaining hapten DXM-2C.
Preferably, the molar ratio of the dextromorphan to the ethyl bromoacetate is 1:1-2.
Further preferably, the molar ratio of dextromorphan to ethyl bromoacetate is 1:1.2
Preferably, the molar ratio of the dextromorphan to the potassium carbonate is 1-1.5:4-6.
Further preferably, the molar ratio of dextromorphan to potassium carbonate is 1:4.
Preferably, the solvent is acetone, methanol or acetonitrile.
Preferably, the concentration of the sodium hydroxide aqueous solution is 1mol/L.
Preferably, the pH is adjusted to 6-7 by using 1mol/L hydrochloric acid solution.
The application of the dextromethorphan hapten in preparing dextromethorphan artificial antigen is also within the protection scope of the invention.
A dextromethorphan artificial antigen, which has a structural formula shown in a formula (II):
Figure BDA0003521971410000033
wherein protein is a carrier protein.
Preferably, the carrier protein is any one or more of 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 dextromethorphan artificial antigen is that the dextromethorphan hapten DXM-2C of the formula (I) is coupled with carrier protein.
Preferably, the carrier protein is coupled by the active ester method.
As a specific embodiment of the method, the preparation method of the dextromethorphan artificial antigen comprises the following steps:
(1) Dissolving DXM-2C and N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring for 2-4 hours at room temperature in a dark place to obtain DXM-2C activating solution;
(2) The carrier protein was added to PBS buffer (0.01 mol/L, pH=7.4);
(3) Slowly and dropwise adding the DXM-2C 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 dextromethorphan artificial antigen.
Preferably, in the step (1), the mass ratio of the DXM-2C, NHS to the EDC is 1-2:1-2:2-3.
More preferably, the mass ratio of DXM-2C, NHS to EDC in step (1) is 1:1.6:2.
Preferably, the ratio of the carrier protein to PBS buffer in step (2) is 10 mg/1 mL.
Preferably, the mass ratio of DXM-2C in step (1) to the carrier protein in step (2) is 1-5:3-8.
More preferably, the mass ratio of DXM-2C in step (1) to the carrier protein in step (2) is 1:4.
Preferably, the dialysis in step (4) is three days, 3 times a day.
The application of the dextromethorphan artificial antigen in preparing dextromethorphan antibodies is also within the protection scope of the invention.
A dextromethorphan antibody is prepared by immunizing an animal with any one of the dextromethorphan artificial antigens.
Preferably, the dextromethorphan antibody is prepared by immunizing an animal with dextromethorphan artificial antigen (DXM-2C-KLH) using a carrier protein as a Keyhole Limpet Hemocyanin (KLH).
Preferably, the dextromethorphan antibody is a monoclonal antibody or a polyclonal antibody.
As a specific embodiment of the above method, the preparation method of dextromethorphan polyclonal antibody comprises the following steps:
(1) The prepared hapten DXM-2C coupled keyhole limpet hemocyanin artificial antigen (DXM-2C-KLH) is used as an immunogen and is uniformly emulsified with an equal amount of immune adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the later booster immunization) 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 rises, the ear margin vein is adopted to strengthen 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.
Dextromethorphan polyclonal antibodies prepared by the method are also within the scope of the invention.
The application of the dextromethorphan artificial antigen and the dextromethorphan antibody in detecting dextromethorphan and/or preparing products for detecting dextromethorphan is also within the protection scope of the invention.
The utility model provides a dextromethorphan colloidal gold short-term test card, includes PVC bottom plate and arranges sample pad, gold mark conjugate pad, nitrocellulose membrane and the pad that absorbs water on the bottom plate in proper order, the specific antibody that colloidal gold marked artificial antigen (DXM-2C-KLH) immune animal preparation obtained is adsorbed in the gold mark conjugate pad, painted quality control line and detection line on the nitrocellulose membrane, the detection line is with the coating antigen solution spraying obtains, the quality control line is with sheep anti-rabbit antibody spraying obtains.
Preferably, the gold-labeled conjugate pad is internally adsorbed with a specific antibody prepared by immunizing an animal with the artificial antigen DXM-2C-KLH marked by colloidal gold.
Preferably, the coating antigen is an artificial antigen DXM-2C-OVA using carrier protein as chicken ovalbumin.
An immunoassay method for detecting dextromethorphan, which uses any one of the dextromethorphan artificial antigens as a coating antigen and any one of the dextromethorphan antibodies as a detection antibody.
Preferably, the specific antibody prepared by immunizing an animal with the artificial antigen DXM-2C-OVA of which the carrier protein is chicken ovalbumin and the artificial antigen DXM-2C-KLH of which the carrier protein is keyhole limpet hemocyanin is used as a coating antigen for detection.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
Compared with the prior art, the invention has the following benefits:
the invention provides a dextromethorphan hapten, which is used for preparing dextromethorphan artificial antigen, further immunizing animals to prepare a specific antibody for detecting dextromethorphan, and the antibody has good specificity and detection sensitivity to dextromethorphan, has the half inhibition concentration of 4.42ng/mL to dextromethorphan, has the detection limit of 0.18ng/mL and the quantitative detection range of 0.59-33.37 ng/mL, has no cross reaction to other structural analogues and functional analogues, and establishes an immunoassay method for detecting dextromethorphan with high sensitivity, stability and rapidness by using the antibody so as to realize the rapid detection of dextromethorphan; meanwhile, the invention also develops a colloidal gold rapid detection kit based on the dextromethorphan artificial antigen and the antibody, which can specifically identify dextromethorphan and has the characteristics of high sensitivity and strong specificity.
Drawings
FIG. 1 is a synthetic route pattern of dextromethorphan hapten (DXM-2C) of example 1 of the present invention.
Fig. 2 is a UV scan of DXM-2C, KLH, DXM-2C-KLH of example 2 of this application.
FIG. 3 is a UV scan of DXM-2C, OVA, DXM-2C-OVA of example 2 of the present application.
FIG. 4 is a UV scan of DXM-2C, LF, DXM-2C-LF of example 2 of the present application.
Fig. 5 is an indirect competition ELISA standard curve for the antibodies to dextromethorphan of example 5 of the present application.
Fig. 6 is a schematic structural diagram of a dextromethorphan colloidal gold rapid detection test strip according to example 8 of the present application; wherein: 1. nitrocellulose membrane (NC membrane); 2. a PVC bottom plate; 3. a sample pad; 4. a gold label conjugate pad; 5. a water absorbing pad; 6. a test line; 7. and a control line.
FIG. 7 is a graph showing the judgment of the result of the test strip for rapid detection of dextromethorphan 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 dextromethorphan hapten
1. Preparation of dextromethorphan hapten DXM-2C
And (3) taking 1mmol of dextromorphan and 4mmol of potassium carbonate as solvents, stirring and reacting with 1.2mmol of ethyl bromoacetate at 40 ℃ for 4-8 hours, separating and purifying reactants through silica gel column chromatography, dissolving the separated and purified reactants in the methanol, adding sodium hydroxide aqueous solution, stirring for 4-6 hours at room temperature, and regulating pH to 6-7 by using hydrochloric acid solution after the reaction is finished, thus obtaining hapten DXM-2C. The synthetic route for DXM-2C is shown in FIG. 1.
2. Identification of dextromethorphan hapten DXM-2C
Nuclear magnetic resonance hydrogen spectrum results of DXM-2C: 1 H NMR(CDCl 3 ,500MHz):12.80(1H,s),6.98(1H,d,J＝5Hz),6.75(1H,d,J＝5Hz),6.69(1H,dd,J＝2.5Hz,1Hz),4.64(2H,s),3.67(1H,s),2.39～2.29(2H,d),2.26(3H,s,J＝15Hz),1.98(1H,dt,J＝10Hz,15Hz),1.75(1H,d,J＝10Hz),1.751(4H,dd),1.60(1H,d,J＝15Hz),1.57～1.31(4H,m),1.31～1.18(1H,m),1.09～0.95(1H,m).
mass spectrometry identification of DXM-2C: MS: C19H25NO3:315.18 ESI+ [ M-H ] +:316.1.
According to the nuclear magnetic resonance hydrogen spectrum and the mass spectrum result, the derivatization site is correct and successful, which shows that the invention successfully synthesizes the target product dextromethorphan hapten DXM-2C, and the structural formula is shown as the formula (I):
Figure BDA0003521971410000071
hapten DXM-2C was named by systematic nomenclature: 2- (((4 bS,8aS, 9S) -11-methyl-6,7, 8a,9,10-hexahydro-5H-9,4b- (epiminoethane-3-yl) oxy) acetic acid, i.e., 2- (((4 bS,8aS, 9S) -11-methyl-6,7, 8a,9,10-hexahydro-5H-9,4b- (iminoethanol) phenanthran-m-3-yl) oxy) acetic acid.
EXAMPLE 2 preparation and identification of dextromethorphan Artificial antigen
1. Preparation of dextromethorphan artificial antigen
Dextromethorphan hapten (DXM-2C) prepared in example 1 was conjugated to Keyhole Limpet Hemocyanin (KLH), chicken Ovalbumin (OVA) and Lactoferrin (LF) by an active ester method;
(1) 10mg of DXM-2C prepared in example 1, 2mg of NHS and 3mg of EDC are respectively weighed and dissolved in 50-100 mu L of DMF, and the mixture is stirred at room temperature for 2-4 hours in a dark place to obtain dextromethorphan hapten activating solution;
(2) 10mg of Keyhole Limpet Hemocyanin (KLH), 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 dextromethorphan 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 three days and 3 times per day, obtaining dextromethorphan artificial antigen (DXM-2C-KLH, DXM-2C-OVA, DXM-2C-LF) after dialyzing, subpackaging in centrifuge tube, and storing at-20deg.C for use.
Wherein, the formula of the phosphate buffer solution comprises: 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 dextromethorphan Artificial antigen
The synthetic dextromethorphan artificial antigen (DXM-2C-KLH, DXM-2C-OVA, DXM-2C-LF) was subjected to ultraviolet full-wavelength scanning, and the results are shown in FIG. 2, FIG. 3, and FIG. 4.
Specifically, KLH and DXM-2C, DXM-2C-KLH were identified by ultraviolet (200-400 nm) scanning, respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of dextromethorphan immunogen DXM-2C-KLH was significantly different from that of carrier protein KLH, DXM-2C had a characteristic peak at 320nm, whereas after coupling reaction, the absorption peak of DXM-2C-KLH was significantly higher than that of KLH at 280nm, and a significant shift was seen by comparing the curves of DXM-2C (FIG. 2). Since the small molecule 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 the complex of carrier protein KLH and DXM-2C, and the coupling of DXM-2C-KLH is successful.
Specifically, OVA and DXM-2C, DXM-2C-OVA were respectively identified by ultraviolet (200-400 nm) scanning, and by comparing the highest absorbance values of the substances before and after coupling, it was found that the absorption curve of the dextromethorphan artificial antigen DXM-2C-OVA was significantly different from that of the carrier protein OVA, DXM-2C had a characteristic peak at 320nm, whereas after coupling reaction, the absorption peak of DXM-2C-OVA was significantly higher than that of OVA at 280nm, and a significant shift was seen by comparing the curves of DXM-2C (FIG. 3). 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 protein OVA and DXM-2C, and the coupling of DXM-2C-OVA is successful.
Specifically, LF and DXM-2C, DXM-2C-LF were respectively identified by ultraviolet (200-400 nm) scanning, and by comparing the highest absorbance values of the substances before and after coupling, the absorption curve of the dextromethorphan artificial antigen DXM-2C-LF was found to be significantly different from that of the carrier protein LF, DXM-2C had a characteristic peak at 320nm, and after coupling reaction, the absorption peak of DXM-2C-LF was significantly higher than LF at 280nm, and a significant shift was found to occur by comparing the curves of DXM-2C (FIG. 4). Since the small molecular components such as unreacted medicines and the like are completely dialyzed and removed in the dialysis process after the coupling, the characteristic peak of the medicines, which appears in the coupled product, is contributed by the medicine molecules combined by the proteins, so that the reaction product is a complex of carrier proteins LF and DXM-2C, and the coupling of the DXM-2C-LF is successful.
Example 3 preparation of antibodies
1. Preparation of polyclonal antibodies
The dextromethorphan artificial antigens DXM-2C-KLH and DXM-2C-LF of DXM-2C-coupled Keyhole Limpet Hemocyanin (KLH) prepared in example 2 are respectively used as immunogens, and are uniformly emulsified with an equal amount of immune adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the subsequent booster immunization) 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
The dextromethorphan artificial antigens DXM-2C-KLH and DXM-2C-LF of the DXM-2C-coupled keyhole limpet hemocyanin KLH prepared in example 2 are respectively used as immunogens, are 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 boost), and are immunized with a Barbish mouse, the mice are immunized by adopting an abdominal subcutaneous multipoint injection method, the serum titer is detected by adopting tail venous blood of the mice after 1 week of boost, and after the antibody titer is no longer increased, the boost is carried out again, and the spleen cells of the mice are taken to fuse with myeloma cells of the mice 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 dextromethorphan immunogen and coating antigen
Screening the coating antigen by using the antibodies obtained after the animals are immunized by taking DXM-2C-KLH and DXM-2C-LF of the example 3 as immunogens respectively through an indirect competition ELISA method, and taking the artificial antigens DXM-2C-KLH, DXM-2C-LF and DXM-2C-OVA prepared in the example 2 as the 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 dextromethorphan immunogen and coating antigen comprising the steps of:
(1) The artificial antigens DXM-2C-KLH, DXM-2C-OVA and DXM-2C-LF prepared in example 2 are respectively used as coating sources, the coating liquid is diluted to 1 mug/mL, 96-well ELISA plates are coated, 100 mug of the coating liquid is added to each well, and the mixture is 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 dextromethorphan polyclonal antibody prepared in example 3 was diluted to seven gradients in a double ratio 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 dextromethorphan 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 BDA0003521971410000101
Figure BDA0003521971410000111
As can be seen from table 1, different dextromethorphan artificial antigens are used as the antibodies generated by the New Zealand white rabbits immunized by the immunogen, and all have certain titers; meanwhile, the obtained antibody has different degrees of inhibition effects on the target analyte dextromethorphan. The potency of the combination of the immunogen and the coating antigen of the number 1 is 1:256000 and the inhibition rate is 88.43 percent, which is higher than the potency and the inhibition rate 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 dextromethorphan, but also have good antibody sensitivity, and DXM-2C-KLH is used as the optimal immunogen, and DXM-2C-OVA is used as the optimal coating antigen.
EXAMPLE 5 establishment of an Indirect competitive ELISA assay for dextromethorphan
1. Experimental method
An indirect competition ELISA method for detecting dextromethorphan comprising the steps of:
(1) The artificial antigen DXM-2C-OVA prepared in example 2 was used as a coating source, diluted to 50. Mu.g/L with a coating solution, coated with 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 6% skimmed milk powder) 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 dextromethorphan polyclonal antibody prepared in example 3 was diluted 16000-fold with PBST, and dextromethorphan standards were 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) Adding 50 mu L of dextromethorphan drug diluent (three groups are parallel) into each row, adding 50 mu L of dextromethorphan polyclonal antibody diluent prepared in the example 3 per hole, incubating for 40min at 37 ℃, washing for 5 times, and beating to dryness;
(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
As shown in FIG. 5, the standard curve of the indirect competition ELISA for detecting dextromethorphan shows that the half inhibitory concentration IC of dextromethorphan by dextromethorphan polyclonal antibody prepared in example 3 is shown in FIG. 5 50 The detection limit is 0.18ng/mL, and the quantitative detection range is 0.59-33.37 ng/mL, which indicates that the antibody for detecting dextromethorphan prepared by the invention can meet the detection requirement and has strong recognition capability on dextromethorphan.
Example 6 evaluation of specificity of antibodies for detection of dextromethorphan
1. Experimental method
The polyclonal antibody prepared in example 3 was tested for cross-reactivity with other structural and functional analogues by changing the drug dextromethorphan in example 5 to dexamethasone, prednisolone, and betametha Mi Songhe fordine and performing the above-described test at the same dilution.
The specificity of the antibody for detecting dextromethorphan is determined by carrying out a cross-reaction experiment on dextromethorphan and structural and functional analogues thereof, wherein the specificity of the antibody is expressed by a cross-reaction rate (CR), and the smaller the cross-reaction rate is, the stronger the specificity is. The dextromethorphan structure and its functional analogues (dexamethasone, prednisolone, betamethasone Mi Songhe Ful can) were diluted by multiple ratio, and measured by indirect competition ELISA method, and the same sensitivity verification method as in example 5 was used to obtain IC of each structural and functional analogue 50 Values, right mesafen cross-reactivity (CR) was calculated according to the following formula:
CR(％)＝IC 50 (dextromethorphan)/IC 50 (structural and functional analogues) ×100%
2. Experimental results
The cross-reaction results of dextromethorphan and its structural and functional analogs are shown in table 2.
TABLE 2 Cross-reaction results of dextromethorphan and its structural and functional analogs
Figure BDA0003521971410000121
Note that: NR indicates no reaction.
As can be seen from table 2, the antibodies used to detect dextromethorphan had no cross-reaction to dexamethasone, prednisolone, betamethasone Mi Songhe forcodeine; the polyclonal antibody prepared in the example 3 for detecting dextromethorphan has strong specificity to dextromethorphan, can effectively eliminate the interference of structural and functional analogues (dexamethasone, prednisolone and beta Mi Songhe Fulgenedine) to dextromethorphan, and can be specially used for detecting dextromethorphan.
EXAMPLE 7 development of ELISA kit for detecting dextromethorphan
1. A kit for detecting dextromethorphan is constructed, the kit comprising the following components:
(1) Preparing an ELISA plate coated with a coating source: the artificial antigen DXM-2C-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 a 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 after the drying; 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) Dextromethorphan 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) Dextromethorphan polyclonal antibody prepared in example 3;
(4) Enzyme conjugate: horseradish peroxidase-labeled dextromethorphan polyclonal antibody prepared in example 3;
(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 graph by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mug/L) of the dextromethorphan 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 dextromethorphan in the sample.
Example 8 development of dextromethorphan colloidal gold rapid test strip
1. Assembly of colloidal gold rapid detection test strip
The colloidal gold rapid detection test strip is formed by superposing a nitrocellulose membrane (NC membrane) 1, a gold-labeled conjugate pad 4, a sample pad 3, a water absorption pad 5 and a PVC bottom plate 2. Specifically, a coating material (DXM-2C-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 6 and a control line 7 (T line and C line) which were located in the middle of the NC film 1 and spaced apart from each other by 7mm, dried at 37℃for 12 hours, then the NC film 1 was stuck on the middle portion of a backing plate, the sample pad 2 was overlapped with the T line 5 end of the NC film by 1mm, the water absorbing pad 5 was stuck on the upper side of the NC film 1 by 1mm, and the assembled test paper plate was cut into test strips 3.50mm wide using a chopper. Fig. 6 is a schematic structural diagram of a dextromethorphan colloidal gold rapid test strip.
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 is regulated to about 8.5, 10 mug of antibody is added for incubation for 30min, then 10% BSA solution is added for incubation for 30min, and then the solution is separated at 10000rpm at 4 DEG CHearts were removed for 20min and the supernatant was resuspended in 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 3 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 dextromethorphan as the test object, 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 test sample is negative (as shown in FIG. 7A); if the sample contains dextromethorphan as an object to be detected, the dextromethorphan 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 dextromethorphan artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling dextromethorphan hapten with keyhole limpet hemocyanin, and the coating antigen is obtained by coupling dextromethorphan hapten with chicken egg white albumin; the structural formula of the dextromethorphan hapten is shown as a formula (I):
Figure QLYQS_1
formula (I).
2. The dextromethorphan artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling dextromethorphan hapten with keyhole limpet hemocyanin, and the coating antigen is obtained by coupling dextromethorphan hapten with lactoferrin; the structural formula of the dextromethorphan hapten is shown as a formula (I):
Figure QLYQS_2
formula (I).
3. The dextromethorphan artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling dextromethorphan hapten with lactoferrin, and the coating antigen is obtained by coupling dextromethorphan hapten with chicken egg white albumin; the structural formula of the dextromethorphan hapten is shown as a formula (I):
Figure QLYQS_3
formula (I).
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FR2658610A1 (en) * 1990-02-16 1991-08-23 Stabiligen Immunoassay of dextromethorphan and/or dextrorphan in biological samples
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US7238791B1 (en) * 2005-12-16 2007-07-03 Roche Diagnostics Operations, Inc. 6-monoacetylmorphine derivatives useful in immunoassay
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