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

Abstract

The present invention provides a dextromethorphan hapten, an artificial antigen, an antibody and a preparation method and application thereof. The structural formula of the dextromethorphan hapten is shown in formula (I), and the dextromethorphan hapten is coupled to a carrier protein Obtain the artificial antigen, and further immunize the animals to prepare the specific antibody for detecting dextromethorphan. The artificial antigen is used as the coating source. The antibody has good specificity and detection sensitivity to dextromethorphan. The inhibitory concentration is 4.42ng/mL, the detection limit is 0.18ng/mL, the quantitative detection range is 0.59-33.37ng/mL, and there is no cross reaction to other structural analogs and functional analogs. The highly sensitive, stable and rapid immunoassay method for detecting dextromethorphan realizes the purpose of rapid detection of dextromethorphan.

Description

A kind of dextromethorphan hapten, artificial antigen, antibody and its preparation method and application

technical field

The invention relates to the technical field of food detection, more specifically, to a dextromethorphan hapten, an artificial antigen, an antibody and a preparation method and application thereof.

Background technique

Dextromethorphan (DXM), also known as dextromethorphan, is the dextro-isomer of morphine-like levomorphan methyl ether. Clinically, it is an efficient and widely used over-the-counter antitussive, which works by inhibiting the medullary cough center, and is mostly used to treat colds, pharyngitis, acute or chronic bronchitis, and coughs caused by upper respiratory tract infections. There is no tolerance and addiction in the therapeutic dose, and the effect is fast and safe. Although it shows significant advantages in clinical treatment, with the widespread use of dextromethorphan, dextromethorphan dependence and abuse gradually appear in the population. Overdose of dextromethorphan can cause euphoria, hallucinations, excitatory impulses and dissociative sedation in the human body, and withdrawal symptoms such as dizziness, anxiety, insomnia and cognitive emotional disturbance will appear after discontinuation, showing obvious drug dependence.

A recent report found that in order to increase the efficacy of herbal teas for greater commercial profits, unscrupulous traders illegally mixed dextromethorphan into herbal teas that claimed to have a strong antitussive effect. The dosage of the illegally added drugs in the herbal tea produced and sold on the spot is arbitrary, mixed in various ways, and the incompatibility is not paid attention to. There is no professional pharmacist's guidance and dosage restrictions when selling. If consumers drink a large amount or for a long time without knowing it, repeated or overdose may occur, which poses a great safety hazard. This illegal behavior seriously violated the Food Safety Law, and at the same time brought potential health threats to consumers without their knowledge.

At present, the detection methods for dextromethorphan mainly include instrumental analysis methods such as high performance liquid chromatography, capillary gas chromatography and liquid chromatography-mass spectrometry, which have the advantages of good accuracy and high sensitivity, but the equipment is expensive and requires The operation by professionals cannot meet the needs of on-site rapid detection, and it is difficult to popularize it at the grassroots level. Therefore, the establishment of a detection method for dextromethorphan in herbal tea that is simple, easy, highly sensitive, and specific is an important technical support for ensuring food safety and is of great significance to safeguarding people's health. Immunization methods are low in cost and easy to operate, and play an irreplaceable role in the field of on-site rapid screening. The key technology is to obtain highly sensitive and specific antibodies. However, there is still a lack of highly sensitive and specific dextromethorphan antibodies and dextromethorphan haptens and artificial antigens that can be used to produce highly sensitive and specific antibodies.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the dextromethorphan detection method in the prior art, and provide a dextromethorphan hapten, artificial antigen, antibody and its preparation method and application.

The object of the present invention is to provide a dextromethorphan hapten.

The object of the present invention is also to provide a preparation method of the dextromethorphan hapten.

The object of the present invention is also to provide the application of the dextromethorphan hapten in the preparation of dextromethorphan artificial antigen.

The object of the present invention is also to provide a dextromethorphan artificial antigen.

The object of the present invention is also to provide a preparation method of the dextromethorphan artificial antigen.

The purpose of the present invention is also to provide the application of the dextromethorphan artificial antigen in the preparation of dextromethorphan artificial antibody.

The purpose of the present invention is also to provide a dextromethorphan antibody.

The object of the present invention is also to provide a kit for detecting dextromethorphan.

The object of the present invention is also to provide an immunoassay method for detecting dextromethorphan.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention provides a hapten DXM-2C of dextromethorphan, the structural formula of the hapten DXM-2C is shown in formula (I):

The hapten DXM-2C is named by systematic nomenclature: 2-(((4bS,8aS,9S)-11-methyl-6,7,8,8a,9,10-hexahydro-5H-9,4b- (epiminoethano)phenan thren-3-yl)oxy)acetic acid, namely 2-(((4bS,8aS,9S)-11-methyl-6,7,8,8a,9,10-hexahydro-5H- 9,4b-(iminoethanol)phenanthrenin-3-yl)oxy)acetic acid.

The preparation method of the hapten DXM-2C of the present invention is to react dextrohydroxymorphan and ethyl bromoacetate in a solvent, separate and purify the reactant, and hydrolyze it in an alkaline environment to obtain the hapten DXM-2C.

The structural formula of the dextromethorphan is:

The structural formula of described ethyl bromoacetate is:

Specifically, dissolve dextrohydroxymorphan, potassium carbonate and ethyl bromoacetate in a solvent, stir, condense and reflux at 40°C for 4-8 hours, separate and purify the reactants, and dissolve the separated and purified reactants in methanol , and then add sodium hydroxide aqueous solution and stir at room temperature for 4-6 hours. After the reaction, adjust the acid with hydrochloric acid solution to obtain the hapten DXM-2C.

Preferably, the molar ratio of dexoxymorphan to ethyl bromoacetate is 1:1-2.

Further preferably, the molar ratio of dextrohydroxymorphan to ethyl bromoacetate is 1:1.2

Preferably, the molar ratio of dexoxymorphan to potassium carbonate is 1-1.5:4-6.

Further preferably, the molar ratio of dextromethorphan to potassium carbonate is 1:4.

Preferably, the solvent is acetone, methanol or acetonitrile.

Preferably, the concentration of the aqueous sodium hydroxide solution is 1 mol/L.

Preferably, the acid adjustment is to adjust the pH to 6-7 with 1 mol/L hydrochloric acid solution.

The application of the dextromethorphan hapten in the preparation of dextromethorphan artificial antigen is also within the protection scope of the present invention.

A dextromethorphan artificial antigen, the structural formula of the dextromethorphan artificial antigen is shown in formula (II):

Wherein, protein is a carrier protein.

Preferably, the carrier protein is bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (Lactoferrin, LF) or chicken ovalbumin (Ovalbumin, OVA ) any one or several.

The preparation method of the dextromethorphan artificial antigen is obtained by coupling a carrier protein to the carboxyl group of the dextromethorphan hapten DXM-2C described in the formula (I).

Preferably, the carrier protein is coupled by the active ester method.

As a specific embodiment of the above method, the preparation method of the dextromethorphan artificial antigen comprises the following steps:

(1) Dissolve DXM-2C, N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in N, In N-dimethylformamide (DMF), stir at room temperature in the dark for 2 to 4 hours to obtain DXM-2C activation solution;

(2) adding carrier protein to PBS buffer (0.01mol/L, pH=7.4);

(3) Slowly add the DXM-2C activation solution in step (1) dropwise to the carrier protein buffer solution in step (2), and react at 4°C for 12 hours;

(4) dialyze the reaction solution obtained in step (3) with PBS buffer solution to obtain the dextromethorphan artificial antigen.

Preferably, the mass ratio of DXM-2C, NHS and EDC in step (1) is 1-2:1-2:2-3.

More preferably, the mass ratio of DXM-2C, NHS and EDC in step (1) is 1:1.6:2.

Preferably, the mass volume 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 performed for three days, 3 times a day.

The application of the dextromethorphan artificial antigen in the preparation of dextromethorphan antibody is also within the protection scope of the present invention.

A dextromethorphan antibody is prepared by immunizing animals with any of the dextromethorphan artificial antigens described above.

Preferably, the dextromethorphan antibody is prepared by immunizing animals with a dextromethorphan artificial antigen (DXM-2C-KLH) whose carrier protein is 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 artificial antigen (DXM-2C-KLH) of the prepared hapten DXM-2C coupled with keyhole limpet hemocyanin (DXM-2C-KLH) was used as the immunogen and the same amount of immune adjuvant (complete Freund's adjuvant for the first immunization) , after boosting immunization with incomplete Freund's adjuvant) emulsified evenly, immunized animals. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles and ear veins respectively. The second immunization was done after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization;

(2) One week after booster immunization, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 10,000 rpm/min at 4°C for 15 minutes, and the supernatant was taken as antiserum. Antisera were purified to polyclonal antibodies by ammonium sulfate precipitation.

The dextromethorphan polyclonal antibody prepared by the above method is also within the protection scope of the present invention.

The application of the dextromethorphan artificial antigen and dextromethorphan antibody in detection of dextromethorphan and/or preparation of products for detection of dextromethorphan is also within the protection scope of the present invention.

A colloidal gold rapid detection card for dextromethorphan, comprising a PVC base plate and sample pads, gold standard conjugate pads, nitrocellulose membranes and water-absorbing pads arranged on the base plate in sequence, colloidal gold is adsorbed in the gold standard conjugate pad The specific antibody prepared by immunizing animals with labeled artificial antigen (DXM-2C-KLH), the quality control line and the detection line are sprayed on the nitrocellulose membrane, and the detection line is obtained by spraying the coating antigen solution. The quality control line was sprayed with goat anti-rabbit antibody.

Preferably, the colloidal gold-labeled artificial antigen DXM-2C-KLH is adsorbed in the gold-labeled conjugate pad and the specific antibody prepared by immunizing animals is absorbed.

Preferably, the coating antigen is an artificial antigen DXM-2C-OVA whose carrier protein is chicken ovalbumin.

An immunoassay method for detecting dextromethorphan, using any of the dextromethorphan artificial antigens as a coating antigen, and using any of the dextromethorphan antibodies as a detection antibody for detection.

Preferably, the specific antibody prepared by immunizing animals with the artificial antigen DXM-2C-OVA whose carrier protein is chicken ovalbumin as the coating antigen and the artificial antigen DXM-2C-KLH whose carrier protein is keyhole limpet hemocyanin to test.

The immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensing, immunocolloidal gold, and the like.

Compared with the prior art, the present invention has the following benefits:

The invention provides a dextromethorphan hapten, using the hapten to prepare an artificial dextromethorphan antigen, and further immunizing animals to prepare a specific antibody for detecting dextromethorphan, using the artificial antigen as a coating source, The antibody has good specificity and detection sensitivity for dextromethorphan, its half-inhibitory concentration for dextromethorphan is 4.42ng/mL, the detection limit is 0.18ng/mL, and the quantitative detection range is 0.59-33.37ng/mL. Structural analogs and functional analogs have no cross-reactivity, and a highly sensitive, stable, and rapid immunoassay method for detecting dextromethorphan has been established by using the antibody, and the purpose of rapid detection of dextromethorphan has been realized; meanwhile, the present invention also develops A colloidal gold rapid detection kit based on the dextromethorphan artificial antigen and antibody has been developed, which can specifically recognize dextromethorphan, and has the characteristics of high sensitivity and strong specificity.

Description of drawings

Fig. 1 is a synthetic route diagram of the dextromethorphan hapten (DXM-2C) of Example 1 of the present invention.

Fig. 2 is the ultraviolet scanning diagram of DXM-2C, KLH, DXM-2C-KLH of Example 2 of the present application.

Fig. 3 is the ultraviolet scanning diagram of DXM-2C, OVA, DXM-2C-OVA of Example 2 of the present application.

Fig. 4 is the ultraviolet scanning diagram of DXM-2C, LF and DXM-2C-LF of Example 2 of the present application.

Fig. 5 is an antibody indirect competition ELISA standard curve of dextromethorphan in Example 5 of the present application.

Fig. 6 is the structural representation of the dextromethorphan colloidal gold rapid detection test strip of the embodiment 8 of the present application; Wherein: 1, nitrocellulose membrane (NC membrane); 2, PVC bottom plate; 3, sample pad; 4, gold standard Conjugate pad; 5. Absorbent pad; 6. Test line; 7. Control line.

Fig. 7 is the result determination diagram of the dextromethorphan colloidal gold rapid detection test strip of Example 8 of the present application; wherein: A is the test result of the negative sample, B is the test result of the positive sample, and C and D are the failure of the test strip.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Preparation and identification of embodiment 1 dextromethorphan hapten

1. Preparation of dextromethorphan hapten DXM-2C

1mmol of dextromethorphan, 4mmol of potassium carbonate, methanol as a solvent, and 1.2mmol of ethyl bromoacetate were stirred and reacted at 40°C for 4 to 8 hours, and the reactant was separated and purified by silica gel column chromatography, and the separated and purified reactant was Dissolve in methanol, then add aqueous sodium hydroxide solution and stir at room temperature for 4-6 hours. After the reaction, adjust the pH to 6-7 with hydrochloric acid solution to obtain the hapten DXM-2C. The synthetic route of DXM-2C is shown in Figure 1.

2. Identification of dextromethorphan hapten DXM-2C

DXM-2C proton nuclear magnetic resonance spectrum results: 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).

The mass spectrum identification result of DXM-2C: MS: C19H25NO3: 315.18, ESI+[M-H]+: 316.1.

According to the proton nuclear magnetic resonance spectrum and mass spectrometry results, it can be seen that the derivation site is correct and successful, indicating that the present invention has successfully synthesized the target product dextromethorphan hapten DXM-2C, and its structural formula is as shown in formula (I):

The hapten DXM-2C is named systematically: 2-(((4bS,8aS,9S)-11-methyl-6,7,8,8a,9,10-hexahydro-5H-9,4b-(epiminoethano )phena nthren-3-yl)oxy)acetic acid, namely 2-(((4bS,8aS,9S)-11-methyl-6,7,8,8a,9,10-hexahydro-5H-9, 4b-(iminoethanol)phenanthrenin-3-yl)oxy)acetic acid.

Example 2 Preparation and Identification of Dextromethorphan Artificial Antigen

1. Preparation of dextromethorphan artificial antigen

The dextromethorphan hapten (DXM-2C) prepared in Example 1 was coupled to keyhole limpet hemocyanin (KLH), chicken ovalbumin (OVA) and lactoferrin (LF) by the active ester method;

(1) Dissolve 10 mg of DXM-2C prepared in Example 1, 2 mg of NHS and 3 mg of EDC in 50-100 μL DF, and stir at room temperature for 2-4 hours in the dark to obtain a dextromethorphan hapten activation solution;

(2) Weigh 10 mg of keyhole limpet hemocyanin (KLH), chicken ovalbumin (OVA) and lactoferrin (LF) into 1 mL of PBS buffer (0.01 mol/L, pH=7.4);

(3) Slowly add the dextromethorphan hapten activation solution obtained in step (1) dropwise to the carrier protein buffer solution obtained in step (2), and stir at 4°C for 12 hours;

(4) Dialyze with PBS buffer solution for three days, 3 times a day, and obtain dextromethorphan artificial antigens (DXM-2C-KLH, DXM-2C-OVA, DXM-2C-LF) after the dialysis, and distribute them in centrifuge tubes , stored at -20°C until use.

Among them, the formula of the phosphate buffer solution: Na ₂ HPO ₄ ·12H ₂ O ₂ .90g, NaCl 8.50g, KCl 0.20g, KH ₂ PO ₄ 0.20g, add distilled water to 1000mL.

2. Identification of dextromethorphan artificial antigen

The dextromethorphan artificial antigens (DXM-2C-KLH, DXM-2C-OVA, DXM-2C-LF) synthesized above were taken for full-wavelength ultraviolet scanning, and the results are shown in Figure 2, Figure 3, and Figure 4.

Specifically, KLH, DXM-2C, and DXM-2C-KLH were identified by ultraviolet (200-400nm) scanning, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the dextromethorphan immunogen DXM-2C-KLH The absorption curve of DXM-2C-KLH is significantly different from that of the carrier protein KLH. DXM-2C has a characteristic peak at 320nm, and after the coupling reaction, the absorption peak of DXM-2C-KLH is significantly higher than that of KLH at 280nm, and compared with that of DXM-2C It can be seen from the curve that a significant displacement has occurred (Fig. 2). Since the unreacted drug and other small molecule components have been dialyzed and removed in the dialysis process after coupling, the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so it shows that the reaction product is the carrier protein KLH and The complex of DXM-2C, DXM-2C-KLH was successfully coupled.

Specifically, OVA, DXM-2C, and DXM-2C-OVA were identified by ultraviolet (200-400nm) scanning, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the dextromethorphan artificial antigen DXM-2C-OVA The absorption curve of DXM-2C-OVA is significantly different from that of the carrier protein OVA. DXM-2C has a characteristic peak at 320nm, and after the coupling reaction, the absorption peak of DXM-2C-OVA is significantly higher than that of OVA at 280nm, and compared with that of DXM-2C The curve can be seen to have a significant displacement (Figure 3). Since the unreacted drug and other small molecule components have been dialyzed and removed in the dialysis process after coupling, the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so it shows that the reaction product is the carrier protein OVA and The complex of DXM-2C, DXM-2C-OVA was successfully coupled.

Specifically, LF, DXM-2C, and DXM-2C-LF were identified by ultraviolet (200-400nm) scanning, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the dextromethorphan artificial antigen DXM-2C-LF The absorption curve of DXM-2C-LF is significantly different from that of the carrier protein LF. DXM-2C has a characteristic peak at 320nm, and after the coupling reaction, the absorption peak of DXM-2C-LF is obviously higher than that of LF at 280nm, and compared with that of DXM-2C It can be seen from the curve that a significant displacement has occurred (Fig. 4). Since the unreacted drug and other small molecule components have been dialyzed and removed in the dialysis process after coupling, the characteristic peak of the drug that appears in the coupling product is contributed by the protein-bound drug molecule, so it shows that the reaction product is the carrier protein LF and The complex of DXM-2C, DXM-2C-LF was successfully coupled.

The preparation of embodiment 3 antibody

1. Preparation of polyclonal antibodies

The dextromethorphan artificial antigen DXM-2C-KLH of the DXM-2C prepared in Example 2 coupled to keyhole limpet hemocyanin (KLH) and the dextromethorphan artificial antigen DXM-2C coupled to lactoferrin (LF) 2C-LF was used as the immunogen, emulsified evenly with the same amount of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations), and immunized animals. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles and ear veins respectively. The second immunization was done after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week after booster immunization, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 4°C and 10,000 rpm/min for 15 minutes, and the supernatant was taken as antiserum. Antiserum was purified by ammonium sulfate precipitation to obtain polyclonal antibodies, which were frozen at -20°C for future use.

2. Preparation of monoclonal antibodies

The dextromethorphan artificial antigen DXM-2C-KLH of DXM-2C prepared in Example 2 coupled to keyhole limpet hemocyanin KLH and the dextromethorphan artificial antigen DXM-2C-LF of DXM-2C coupled to lactoferrin LF were respectively As an immunogen, emulsify evenly with the same amount of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations), and immunize Barbizi mice with multi-point subcutaneous tissue in the abdomen. Mice were immunized by injection. One week after each booster immunization, blood from the tail vein of the mice was collected to detect the serum titer. After the antibody titer no longer increased, a booster immunization was carried out again. Seven days later, the splenocytes of the mice and small Mouse myeloma cells were fused. The hybridoma cells were screened out in HAT medium and cultured in complete medium. The cell supernatant was detected by the ic-ELISA method, and the cells in the wells with strong positive results were cloned and cultured by the limiting dilution method. After one week, the cells were tested again, the wells were picked, and cloned again. After 3 clone culture tests, the hybridoma cells of the monoclonal antibody were obtained. After the hybridoma cells are amplified and cultured, they are inoculated into the peritoneal cavity of mice to produce ascites containing antibodies. Ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain monoclonal antibodies, which were frozen at -20°C for future use.

The screening of embodiment 4 dextromethorphan immunogen and coating former

Using the DXM-2C-KLH and DXM-2C-LF of Example 3 as immunogens to immunize the antibodies obtained from animals, the coating agent was screened by the indirect competition ELISA method, and the artificial antigen DXM-2C-LF prepared in Example 2 2C-KLH, DXM-2C-LF and DXM-2C-OVA were used as coating agents; the best immunogens and coating agents were selected through the serum titer and inhibition rate obtained by indirect competition ELISA method.

An indirect competitive ELISA method for screening dextromethorphan immunogen and coating agent, comprising the following steps:

(1) The artificial antigens DXM-2C-KLH, DXM-2C-OVA, and DXM-2C-LF prepared in Example 2 were respectively used as coating sources, diluted to 1 μg/mL with coating solution, and coated with 96-well enzyme labels plate, add 100 μL to each well, and incubate at 37°C overnight (12h);

(2) Discard the coating solution, wash twice, and pat dry;

(3) Add 120 μL of blocking solution (i.e. 1% fish skin collagen) to each well, and block at 37°C for 3 hours;

(4) Discard the blocking solution, clap, dry at 37°C for 30 minutes and take it out;

(5) Dilute the dextromethorphan polyclonal antibody prepared in Example 3 into seven gradients with PBST, namely 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, set blank control hole (replaced with PBST) simultaneously; And dilute dextromethorphan medicine to 1 μ g/mL;

(6) Titer column: add 50 μL of PBST to each well, and then add the antibody obtained by doubling dilution to the wells in order of 50 μL per well. No antibody is added to the last well, and 50 μL of PBST is used instead;

Inhibition column: add 50 μL of drug to each well, and then add the antibody obtained by doubling dilution into the wells in turn according to 50 μL per well. No antibody is added to the last well, and 50 μL of PBST is used instead; incubate at 37°C for 40 minutes, wash 5 times, shoot Dry;

(7) Add goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μL/well, incubate at 37°C for 30 minutes, wash five times, and pat dry;

(8) Add color developing solution, 100 μL per well, develop color for 10 min;

(9) Add 50 μL of 2 mol/L H ₂ SO ₄ solution to terminate the reaction, and read the OD value at 450 nm.

The titer is the antibody dilution corresponding to an OD ₄₅₀ of around 1.0.

Inhibition rate=(OD value of potency-OD value of inhibition)/OD value of inhibition*100%.

Table 1 shows the results of serum titers and inhibition rates of different combinations of immunogens and coating agents.

Table 1 Serum titer and inhibitory rate of different immunogens and coating original combinations

As can be seen from Table 1, the antibodies produced by New Zealand white rabbits immunized with different dextromethorphan artificial antigens as immunogens all have certain titers; at the same time, the obtained antibodies have different degrees of inhibition on the target analyte dextromethorphan Effect. Among them, the titer 1:256000 and inhibition rate 88.43% shown by the combination of immunogen and coating source No. 1 are higher than the titer and inhibition rate of combination of immunogen and coating source No. 2, 3, 4, by This can explain that the immunogen and coating agent No. 1 can not only specifically recognize the target analyte dextromethorphan, but also have good antibody sensitivity, so DXM-2C-KLH is used as the best immunogen and DXM-2C-OVA as the best immunogen. Good quilt original.

The establishment of the indirect competition ELISA detection method of embodiment 5 dextromethorphan

1. Experimental method

An indirect competitive ELISA method for detecting dextromethorphan, comprising the following steps:

(1) Use the artificial antigen DXM-2C-OVA prepared in Example 2 as the coating source, dilute it to 50 μg/L with the coating solution, coat the 96-well microtiter plate, add 100 μL to each well, and incubate overnight at 37°C ( 12h);

(2) Discard the coating solution, wash twice, and pat dry;

(3) Add 120 μL of blocking solution (6% skimmed milk powder) to each well, and block at 37°C for 3 hours;

(4) Discard the blocking solution, clap, dry at 37°C for 30 minutes and take it out;

(5) Dilute the dextromethorphan polyclonal antibody prepared in Example 3 by 16000 times with PBST, and dilute the dextromethorphan standard to 1000 μg/L, 200 μg/L, 40 μg/L, 8 μg/L, 1.6 μg/L , 0.32μg/L, 0.064μg/L, 0.0128μg/L;

(6) Add 50 μL of dextromethorphan drug dilution solution to each row (three groups in parallel), then add 50 μL/well of the dextromethorphan polyclonal antibody dilution solution prepared in Example 3, incubate at 37°C for 40min, wash 5 times, and shoot Dry;

(7) Add goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μL/well, incubate at 37°C for 30 minutes, wash five times, and pat dry;

(8) Add color developing solution, 100 μL per well, develop color for 10 min;

(9) Add 50 μL of 2 mol/L H ₂ SO ₄ solution to terminate the reaction, and read the OD value at 450 nm.

2. Experimental results

The antibody indirect competition ELISA standard curve used to detect dextromethorphan is shown in Figure 5. From Figure 5, it can be seen that the half-inhibitory concentration IC of the dextromethorphan polyclonal antibody prepared in Implementation 3 to _{dextromethorphan} is 4.42ng/mL, and the detection The limit is 0.18ng/mL, and the quantitative detection range is 0.59-33.37ng/mL, indicating that the antibody prepared by the present invention for detecting dextromethorphan can meet the detection requirements, and has a strong ability to recognize dextromethorphan.

Example 6 is used to detect the specificity evaluation of the antibody of dextromethorphan

1. Experimental method

The drug dextromethorphan in embodiment 5 is changed into dexamethasone, prednisolone, betamethasone and pholcodine, and carry out above-mentioned test with the same dilution multiple, measure the polyclonal antibody that embodiment 3 prepares to Cross-reactivity rates with other structural and functional analogues.

The specificity for detecting dextromethorphan is determined by conducting cross-reaction experiments with dextromethorphan and its structural and functional analogues. The specificity of the antibody is expressed by the cross-reaction rate (CR). The smaller the cross-reaction rate, the stronger the specificity . The dextromethorphan structure and its functional analogs (dexamethasone, prednisolone, betamethasone and pholcodine) are respectively diluted in multiples, and the indirect competition ELISA method is used for determination, and the steps are the same as the sensitivity of Example 5 Verify the method, obtain the _IC50 value of each structural and functional analogue, and calculate the dextromethorphan cross-reactivity rate (CR) according to the following formula:

CR(%)=IC ₅₀ (dextromethorphan)/IC ₅₀ (structural and functional analogs)×100%

2. Experimental results

The cross-reactivity results of dextromethorphan and its structural and functional analogues are shown in Table 2.

Table 2 Cross-reactivity results of dextromethorphan and its structural and functional analogues

Note: NR means no response.

As can be seen from Table 2, the antibody that is used to detect dextromethorphan has no cross-reaction to dexamethasone, prednisolone, betamethasone and pholcodine; The cloned antibody has strong specificity to dextromethorphan, and can effectively exclude the interference of its structural and functional analogues (dexamethasone, prednisolone, betamethasone and pholcodine) on dextromethorphan, and can be specifically used For the detection of dextromethorphan.

Embodiment 7 detects the development of the ELISA kit of dextromethorphan

1, set up the test kit that detects dextromethorphan, and described test kit comprises following each part:

(1) Preparation of an ELISA plate coated with a coating source: the artificial antigen DXM-2C-OVA prepared in Example 2 was used as a coating source, and the coating buffer was used to dilute the coating source to 50 μg/L. Add 100 μL to each well, incubate overnight at 37°C in the dark, pour off the liquid in the well, wash with washing solution twice for 30 seconds each time, pat dry, then add 200 μL of blocking solution to each well, incubate at 25°C in the dark for 2 hours, pour off The liquid in the hole is patted dry, and sealed with an aluminum film after drying; the coating buffer is a carbonate buffer with a pH value of 9.6 and 0.05mol/L, and the blocking solution is a pH value of 7.1 to 7.5, containing 1% to 3% casein, 0.1-0.3mol/L phosphate buffer;

(2) Dextromethorphan standard solution: 8 concentration gradients, respectively 1000μg/L, 200μg/L, 40μg/L, 8μg/L, 1.6μg/L, 0.32μg/L, 0.064μg/L, 0.0128μg /L;

(3) Dextromethorphan polyclonal antibody prepared in Example 3;

(4) Enzyme conjugate: the dextromethorphan polyclonal antibody prepared in Example 3 labeled with horseradish peroxidase;

(5) Substrate chromogenic solution: composed of A liquid and B liquid, A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

(6) The stop solution is 2mol/L H ₂ SO ₄ ;

(7) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01% to 0.03% sodium azide preservative, and 0.1 to 0.3mol/L phosphate buffer, and the percentage is weight volume percentage.

2. Actual sample testing

Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Dilute the enzyme conjugate concentrated solution with the enzyme conjugate diluent according to the required amount at a volume ratio of 1:10 (that is, one part of the enzyme conjugate concentrated solution is added to 10 parts of the enzyme conjugate diluent, ready for immediate use). Add 50 μL of the standard/sample to the corresponding microwell, then add 50 μL of the enzyme conjugate working solution, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 25°C for 30 minutes. Dry the liquid in the well, and add 250 μL/well of washing working solution. Fully wash 4 to 5 times with an interval of 10s each time, pour off the washing solution in the wells of the plate, and pat dry with absorbent paper (after pat dry, the bubbles that are not clear can be punctured with an edible unused pipette tip). Add 50 μL/well of substrate chromogenic solution A, then add 50 μL/well of substrate chromogenic solution B, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 25°C for 10 minutes. The addition is terminated 50 μL/well, shake gently to mix, set the microplate reader to 450nm, and measure the OD value of each well.

3. Analysis of test results

The percent absorbance of a standard or sample is equal to the average of the absorbance values of the standards or samples (dual wells) divided by the average of the absorbance values of the first standard (0 standard), multiplied by 100%. Draw a standard curve with the percent absorbance of the standard as the ordinate and the logarithm of the standard concentration of dextromethorphan (μg/L) as the abscissa. Substitute the percent absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to obtain the actual concentration of dextromethorphan in the sample.

Embodiment 8 Development of Dextromethorphan Colloidal Gold Rapid Detection Test Strip

1. Assembly of colloidal gold rapid detection test strip

The colloidal gold rapid detection test strip is composed of a nitrocellulose membrane (NC membrane) 1, a gold standard conjugate pad 4, a sample pad 3, an absorbent pad 5 and a PVC bottom plate 2. Specifically, the coating agent (DXM-2C-OVA) and goat anti-rabbit IgG were sprayed on the NC film at a spray volume of 0.8 μL/cm with an XYZ three-dimensional spray dot film apparatus, and used as test line 6 and control line 7 (T line and C line), the two lines are located in the middle of the NC film 1 and are separated from each other by 7mm. After drying at 37°C for 12 hours, paste the NC film 1 on the middle part of the liner. The sample pad 2 and the NC film T line 5 The end overlaps by 1mm, and the absorbent pad 5 is pasted on the upper side of the NC film 1 and overlaps it by 1mm. The assembled test paper board is cut into 3.50mm wide test paper strips with a cutter. Fig. 6 is a structural schematic diagram of a rapid detection test strip for dextromethorphan colloidal gold.

2. Preparation of gold-labeled antibody

A colloidal gold suspension with an average diameter of 30 nm was prepared by reducing chloroauric acid with trisodium citrate. Take 1mL colloidal gold solution, add 0.2mol/L _K2CO3 solution to adjust the pH to about 8.5, add 10μg antibody for incubation for 30min, then add 10% BSA solution for incubation for 30min, then centrifuge at 10000rpm at 4° _C for 20min, and remove the supernatant. Resuspend with 200 μL 0.2 mol/L phosphate buffer solution (containing 0.5% Tween-20, 0.5% BSA, 5% sucrose, 0.3% PVP, 0.03% procline-300) with pH 7.4, and store at 4°C.

3. Detection of herbal tea samples

(1) Extraction and purification of herbal tea samples

Take 0.5mL of herbal tea, add 4.5mL of 0.2mol/L pH 7.4 phosphate buffer solution for dilution, vortex and mix for 30s, and use it as the test solution.

(2) Detection steps

Take 150 μL of the sample to be tested and add it to the microwell, add 3 μL of the gold-labeled antibody to mix evenly by pipetting repeatedly, incubate at room temperature for 5 minutes, then insert the test strip into the microwell and react for 3 minutes, then take out the test strip to remove the sample pad, and judge the result .

(3) Judgment of test results

As shown in Figure 7, if the sample does not contain the analyte dextromethorphan, the gold-labeled antibody binds to the coating on the test line (T line) on the rapid detection strip, making the test line display a clear red The line indicates that the test sample is negative (as shown in Figure 7A); if the sample contains dextromethorphan to be tested, then dextromethorphan will bind to the gold-labeled antibody and cannot be captured by the test line on the rapid test strip, and then test If the line does not develop color, it is positive (as shown in Figure 7B); similarly, the gold-labeled antibody also binds to the goat anti-rabbit IgG on the quality control line (C line) on the cellulose membrane, making the quality control line appear red, and the quality control line is red. The presence or absence of the color of the line indicates whether the test strip is valid or invalid (as shown in Figure 7A, B is valid, C is invalid, and D is invalid). 3 to 5 minutes to determine the test results.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all 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):

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):

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):

formula (I).

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