CN111592468A - Diazepam hapten, diazepam antigen and diazepam antibody as well as preparation method and application thereof - Google Patents
Diazepam hapten, diazepam antigen and diazepam antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a diazepam hapten, an artificial antigen, an antibody, and preparation and application thereof. The diazepam hapten has a molecular structure shown as a formula I
(formula I), the diazepam artificial antigen has the formula IIMolecular structure of (1)
Description
Technical Field
The invention belongs to the technical field of food safety immunodetection, and particularly relates to a hapten, an artificial antigen and an antibody of diazepam of fishes, and a preparation method and application thereof.
Background
With the increasing concern and appeal of people on food safety, the residue of drugs in food has become a social hotspot problem. The diazepam of the fish as a fishing anesthetic can reduce the metabolic strength in the fish body, lead the fish to slow down and enter a state similar to dormancy, thereby preventing the fish from being damaged due to violent movement in a container, reducing the death rate of the fish in the long-distance transportation process and reducing the economic loss. Because of the advantages of easy water solubility, quick effect, short recovery time and the like, the diazepam of the fishes is widely used as a fish anesthetic in the circulation link at present and is the only anesthetic approved by the food and drug administration of the United states at present and can be used for eating the fishes. In addition to the United states, countries such as the European Union and Canada also permit stabilization of fish as a narcotic for fish, but the United states limits the drug holiday to 21 days, the maximum residual quantity (MRL) to 1 mug/mL, and the Canada drug holiday to 5 days, and all have strict use standards. Therefore, the analytical method for rapidly and accurately detecting the stable residual level of the fish in the aquatic product is established, and has great significance for standardizing stable use of the fish and ensuring the safety of the aquatic product.
At present, the monitoring of the diazepam of the fishes mainly depends on instrument analysis methods, such as high performance liquid chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry and liquid chromatography. Although the detection results are accurate and reliable, the methods rely on expensive instruments, are time-consuming and labor-consuming, have high operation requirements, and are not suitable for large-batch rapid screening, so that a rapid and simple detection method for the stabilization of the fishes needs to be invented. The immunoassay based on antigen-antibody specific binding has the characteristics of simple and convenient sample pretreatment, simple operation, rapidness, sensitivity, low detection cost and the like, is known as the rapid detection technology which has the most competition and challenge in the 21 st century, and has wide application prospect in the field of food safety. At present, no research report about the diazepam immunodetection of the fishes is found.
Disclosure of Invention
The invention provides a piscine hapten, a corresponding artificial antigen and antibody thereof and a preparation method thereof aiming at the defect of piscine stability detection in the prior art.
The invention also aims to provide application of the hapten, the artificial antigen and the antibody.
Another objective of the present invention is to provide an immunoassay method for detecting diazepam in fish.
The above object of the present invention is achieved by the following means.
In a first aspect, a piscine diazepam hapten having a molecular structure of formula I:
formula I.
The preparation method of the diazepam hapten comprises the following steps: after the condensation reaction of the diazepam and p-aldehyde benzoic acid in methanol, the diazepam hapten with the molecular structure shown in the formula I is obtained after sodium borohydride reduction.
Specifically, the preparation method of the diazepam hapten comprises the following steps:
s11, dissolving the diazepam in a proper amount of methanol;
s12, adding 1-2 mL of triethylamine after the S11 is dissolved;
s13, dissolving phenylaldehydic acid with the molar ratio equal to that of S11 in a proper amount of methanol, and slowly dripping the solution into S1 under the stirring condition;
s14, carrying out condensation reflux reaction for 2-3h at 80 ℃;
s15, adding 0.12-0.2 g of sodium borohydride for reduction, and carrying out condensation reflux at 50 ℃ for 1-2 h;
s16, carrying out rotary evaporation to obtain a yellow oily substance, dissolving the yellow oily substance in water, extracting the yellow oily substance for 3 times by using ethyl acetate, and carrying out rotary drying on an ethyl acetate extract to obtain a light yellow powder;
s17, passing the light yellow powder through a silica gel column, eluting with petroleum ether and ethyl acetate =3:1, and evaporating to dryness to obtain the FISH stable hapten shown in the formula I.
In a second aspect, a piscine-stable artificial antigen has the following molecular structure:
formula II
Wherein the carrier protein is bovine serum albumin or chicken egg albumin.
The invention reserves the space structure of the fish diazepam, the hapten derives an arm containing a benzoic acid structure on the basis of the original molecule, and the antigen of the invention can obtain high-quality and high-efficiency antibody after animal immunization.
Because the quantity limit standard of the diazepam per se is less, the synthesis of hapten and artificial antigen is also rarely reported, and related immunodetection methods are also less, while the existing diazepam detection technology mainly takes an instrument method as a main method, and the instrument method is complex in operation, high in pretreatment requirement and high in cost. The titer of antiserum obtained by immunizing animals by using the artificial antigen can reach 4K, the minimum detection limit is 28.68 ng/mL, the half-inhibitory concentration is 337.84ng/mL, and the antibody has the remarkable advantages of high specificity, good sensitivity, high accuracy and the like.
The preparation method of the diazepam artificial antigen is a carbodiimide method, and preferably comprises the following steps:
s21, respectively dissolving 100-140 mg of the diazepam hapten in 0.2-1 mL of N, N-Dimethylformamide (DMF) to obtain a solution I and a solution II;
s22, respectively dissolving the proteins (50 mg BSA and 50mg OVA) in 10mL of 0.01M PBS buffer solution to obtain a PBS solution of BSA and a PBS solution of OVA; respectively dropwise adding the solution I and the solution II prepared in the step S21 into the PBS solution and the OVA-PBS solution of BSA under stirring, and magnetically stirring for 10 min;
s23, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) with the molar mass of 1.2-1.5 times of the molar mass of the diazepam hapten into the solution obtained in the step S22, and reacting overnight at room temperature in a dark place;
and S24, after the reaction of the S23 solution is finished, dialyzing the solution by PBS to obtain the diazepam artificial antigen shown as the formula II.
As an implementation improvement mode of the preparation method of the diazepam artificial antigen provided by the invention, the pH value of the PBS buffer solution is 7.4-8.0.
In an embodiment of the method for preparing the diazepam artificial antigen, the protein is bovine serum albumin or chicken egg albumin.
In a third aspect, an antibody is prepared from the piscine diazepam artificial antigen. The antibody may be a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.
In a fourth aspect, the artificial antigen or the antibody is used in a fish stable immunodetection assay.
Preferably, the carrier protein for immunogen is artificial antigen of bovine serum albumin, and the carrier protein for coating antigen is artificial antigen of chicken ovalbumin.
In a fifth aspect, an immunoassay method for directly detecting diazepam in fish comprises the following steps:
s31, immunizing animals with the artificial antigen of diazepam to prepare a diazepam polyclonal antibody, wherein the carrier protein is bovine serum albumin;
s32, coating the diazepam artificial antigen serving as a coating antigen on a microporous plate, wherein a carrier protein is egg albumin, and then adding the polyclonal antibody of diazepam prepared in the step S31 into the microporous plate;
s33, adding a sample to be detected, and determining the content of the diazepam in the sample to be detected by adopting competitive ELISA.
Compared with the prior art, the invention has the beneficial effects that:
the titer of antiserum obtained by immunizing animals by using the artificial antigen can reach 1:4000, the minimum detection is 8.68ng/mL, the half-inhibitory concentration is 337.84ng/mL, and the antibody has the remarkable advantages of high specificity, good sensitivity, high accuracy, high detection speed and the like, so that the antigen and the antibody provided by the invention can be used for establishing an enzyme-linked immunosorbent assay technology for the diazepam of fish, can be used for quickly detecting the residual diazepam in food, and have wide application prospects.
Drawings
FIG. 1 is a figure of mass spectrometric identification of diazepam hapten.
FIG. 2 is an ultraviolet scan of the diazepam hapten and its immunizing antigen and carrier protein.
FIG. 3 is a graph showing inhibition curves of antibody prepared from hapten on diazepam in fish.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the examples are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The materials and reagents used in the examples of the present invention are commercially available.
Example 1 preparation of diazepam hapten MS-299
This example provides a method for preparing a piscine hapten MS-299, the molecular structure of the piscine hapten is shown in the following formula I:
formula I.
A method for preparing diazepam hapten MS-299 specifically comprises the following steps:
s11. dissolving 825mg (5 mmol) of piscine in 25mL of anhydrous methanol in a 25mL round-bottom flask;
s12, adding 1mL of triethylamine after dissolution, and magnetically stirring;
s13, slowly dripping 670mg of 4-formylbenzoic acid under magnetic stirring;
s, carrying out condensation reflux reaction for 3 hours at 14.80 ℃;
s15, adding 0.12g of sodium borohydride for reduction, and carrying out condensation reflux reaction at 50 ℃ for 1 h;
s16, spin-drying methanol, dissolving with water, extracting with ethyl acetate for 3 times, and spin-drying ethyl acetate to obtain light yellow powder;
s17, purifying the light yellow powder by a silica gel column, eluting with petroleum ether and ethyl acetate =3:1, and then spin-drying to obtain the product, namely the FISCLE hapten shown in the molecular structure of the formula I.
As shown in fig. 1, the structural analysis of the diazepam hapten is as follows:1H NMR (600 MHz, DMSO) 7.81(d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 7.20 – 7.06 (m, 3H), 6.79–6.77(m, 1H), 6.61 (t, J = 6.0 Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 4.24 (q, J =7.1, 2H), 1.27 (t, J = 7.1 Hz, 3H)。
EXAMPLE 2 preparation of immunogen/coatingen
The immunogen and the coating antigen are prepared by different methods in the use type of carrier protein, wherein the immunogen carrier protein mainly adopts Bovine Serum Albumin (BSA), the coating antigen carrier protein mainly adopts egg white protein (OVA), and the coupling method is a carbodiimide method.
The molecular structure of the immunogen/coatingen is shown in the following formula II,
in the formula II, the compound is shown in the specification,
in the formula, the immune source protein carrier is bovine serum albumin, and the coating source protein carrier is chicken egg albumin.
Taking the preparation method of the immunogen as an example, the method specifically comprises the following steps:
s21, dissolving 140mg of the diazepam hapten (MS-299) prepared in the example 1 in DMF to obtain a hapten dissolved solution;
s22, dissolving 50mg BSA in 10mL PBS buffer solution with pH7.4, adding the hapten dissolving solution obtained in the step S21 under the condition of stirring, and stirring uniformly for about 10 min;
s23, dissolving 90mg of EDC in 200 mu L of water, stirring, dropwise adding into the protein-hapten mixed solution prepared in the step S22, and coupling at room temperature overnight;
s24, the solution in the step 23 reacts to obtain a coupling mixture, and the coupling mixture is dialyzed by PBS for 3 days at 4 ℃ to obtain the fish diazepam immunogen shown as the molecular structure of the formula II.
Respectively diluting the immunogen and the coating antigen to 1mg/mL, subpackaging in a centrifuge tube, and storing at-20 ℃ for use.
Ultraviolet scanning measurement (200-400 nm) is carried out on carrier protein BSA, hapten-piscine-299 and corresponding immunogen thereof, and the characteristic absorption peak of the immunogen is found to shift to the characteristic absorption peak position of the hapten (figure 2), which indicates that the coupling of the immunogen is successful.
Example 3 antibody preparation and characterization
The prepared immunogen and an equal amount of immunologic adjuvant (complete Freund adjuvant is used for the first immunization, and incomplete Freund adjuvant is used for the subsequent boosting immunization) are evenly emulsified to immunize animals. The BALB/c mice of 5-6 weeks are immunized by adopting a plurality of injection modes of back subcutaneous injection, sole subcutaneous injection and abdominal cavity respectively, the second immunization is carried out after 2 weeks, and the boosting immunization is carried out once every 2 weeks later. After the third booster immunization, the mice were bled by tail-off, and the serum titer was determined by indirect competitive ELISA. When the titer no longer increased, intraperitoneal injection was used to boost the immunity. Taking blood from the heart after one week, carrying out water bath for 0.5-1 h, centrifuging at 4 ℃ and 10000 for 15min, and taking the supernatant to obtain the antiserum. The antiserum is purified by an ammonium sulfate precipitation method to obtain a polyclonal antibody, and the polyclonal antibody is frozen at the temperature of minus 20 ℃ for standby.
The result of indirect competition ELISA determination of the positive titer of the antibody is based on the determination value which is 2.1 times that of negative blood, and the result shows that the titer of antiserum (FISTABIN-299-BSA) corresponding to the hapten MS-299 is 1: 4000.
Example 4 specificity and sensitivity of antibodies
According to the above effect, an enzyme-linked immunosorbent assay (ELISA) standard curve is drawn by using antiserum (FIDAM-299-BSA); carbonate buffer (CBS, pH = 9.0) was used as a diluent of the coating source, phosphate buffer (PBS, 0.01 mol/L, pH = 7.4) was used as a diluent of the primary antibody, tween phosphate buffer (PBST, 0.01 mol/L) was used as a diluent of the secondary antibody, MS-299-OVA was used as a coating source: 50 mu L of pharmaceutical standard substance with serial concentration and 50 mu L of FISTABIN multispecific antibody with proper dilution are added into a 96-hole enzyme label plate, and after competitive reaction, the light absorption value (OD) is measured by an enzyme label analyzer. And (3) performing curve fitting on the function by using four parameters of origin 8.5 software by taking the OD value as a vertical coordinate and the corresponding log value of the concentration of the standard as a horizontal coordinate: y = (a-D)/[1+ (X/C) B ] + D where a and D represent the absorbance (OD) of the drug concentration minimum and maximum, respectively, C is the midpoint concentration, the OD value when the standard concentration is equal to C is (a + D)/2, just at the inflection point of the curve, the half maximal inhibitory concentration is IC50, B represents the steepness of the curve, called slope factor: the IC10 is used as the detection limit, and the IC 20-IC 80 are used as the detection range. The standard curve of ELISA was established using diazepam as standard, and the results are shown in FIG. 3, and the relevant standard curve parameters are shown in Table 1. As can be seen by combining the attached drawings and the attached tables 1 and 2, the standard curve established by taking the diazepam as the standard substance has a typical S-shaped curve and good detection sensitivity. Since the specific antibody can directly identify the diazepam, the method can directly measure the content of the diazepam in the food.
TABLE 1 detection parameters of the antiserum obtained by MS-299-BSA immunization on diazepam in fish
| Immunogens | IC50(ng/mL) | Linear range (ng/mL) | Minimum detection limit (ng/mL) | Correlation coefficient |
| MS-299-BSA | 337.84 | 71.26-1601.58555 | 28.68 | 0.99708 |
TABLE 2 Cross-reactivity of antisera raised against diazepam and its structural analogues by MS-299-BSA immunization
| Competitive drugs | IC50(ng/mL) | Rate of cross reaction |
| Fish stabilizer | 337.84 | 100% |
| 4-Aminobenzoic acid ethyl ester | 1212.05 | 27.87% |
| Eugenol | 3633.76 | 0.09% |
| Benzoic acid | >30000 | <0.1% |
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A piscine diazepam hapten, characterized by having the structure shown in the following
Formula I.
2. A process for the preparation of a diazepam hapten according to claim 1, comprising the steps of: after the condensation reaction of the diazepam and p-aldehyde benzoic acid in methanol, the diazepam hapten with the molecular structure shown in the formula I is obtained by sodium borohydride reduction.
3. The method for preparing a diazepam hapten according to claim 2, comprising the steps of:
s11, dissolving the diazepam in a proper amount of methanol;
s12, adding triethylamine after S11 is dissolved;
s13, dissolving phenylaldehydic acid with the molar ratio equal to that of S11 in a proper amount of methanol, and slowly dripping the solution into S1 under the stirring condition;
s14, condensing and refluxing at a preset temperature;
s15, adding sodium borohydride for reduction, and condensing and refluxing at a preset temperature;
s16, carrying out rotary evaporation to obtain a yellow oily substance, dissolving the yellow oily substance in water, extracting the yellow oily substance with ethyl acetate, and carrying out rotary drying on an ethyl acetate extract to obtain light yellow powder;
s17, passing the light yellow powder through a silica gel column, eluting, and then evaporating to dryness to obtain the FISH stable hapten shown in the formula I.
4. A piscine diazepam artificial antigen having a molecular structure represented by formula II:
in the formula II, the compound is shown in the specification,
wherein the carrier protein is bovine serum albumin or chicken egg albumin.
5. A method for preparing a diazepam artificial antigen according to claim 4, comprising the steps of: dissolving the FISH stable hapten as described in claim 1 in DMF, dissolving the carrier protein, adding, stirring uniformly, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and coupling at room temperature overnight; dialyzing the coupling mixture at 4 ℃ with PBS to obtain a diazepam artificial antigen; wherein the molar ratio of the diazepam hapten to the carrier protein is 400-600: 1.
6. the method for preparing a diazepam artificial antigen according to claim 5, which comprises the following steps:
s21, dissolving the carrier protein in a PBS buffer solution to obtain a protein solution;
s22, dissolving the diazepam hapten of the fish as the claim 1 in N, N-dimethylformamide, adding the mixture into the protein solution obtained in the S21, and stirring and mixing to obtain a protein-hapten mixed solution;
s23, dissolving EDC in water, stirring and dripping into the protein-hapten mixed solution of S22, and coupling overnight at room temperature in a dark place;
and S24, after the reaction is finished, performing dialysis by using PBS to obtain the diazepam artificial antigen.
7. An antibody prepared using the piscine-stable artificial antigen of claim 2.
8. The antibody of claim 7, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a genetically engineered antibody.
9. Use of the artificial antigen of claim 2 or the antibody of claim 7 in a fish neuroleptic immunoassay.
10. An immunoassay method for detecting diazepam in fish, which is characterized by comprising the following steps:
s31, immunizing an animal with the diazepam artificial antigen of claim 2 to prepare diazepam polyclonal antibody, wherein the carrier protein is bovine serum albumin;
s32, coating the fish stable artificial antigen as a coating source of claim 2 on a microplate, wherein the carrier protein is chicken ovalbumin; adding the polyclonal antibody of the diazepam prepared in the step S31 into a microplate;
and S33, adding the sample to be detected, and determining the content of the diazepam in the sample to be detected by adopting competitive ELISA.
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