CN114507185B - Phenylbutazone hapten, artificial antigen, antibody and preparation method and application thereof - Google Patents
Phenylbutazone hapten, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a phenylbutazone hapten, an artificial antigen, an antibody and a preparation method and application thereof, wherein the structural formula of the phenylbutazone hapten is shown as a formula (I), the artificial antigen and the antibody for detecting phenylbutazone are prepared by using the hapten, the antibody has high sensitivity and high specificity recognition capability on phenylbutazone, the half inhibition concentration of phenylbutazone is 3.40ng/mL, the detection range is 0.21-53.64 ng/mL, the minimum detection limit is 0.04ng/mL, and the phenylbutazone antibody has no cross reaction on other structural and functional analogues such as piroxicam, ibuprofen, aminopyrine, indomethacin and the like, so that the prepared phenylbutazone antibody has high detection sensitivity and high specificity on phenylbutazone and can meet the field detection requirement on mass samples; in addition, the hapten BTS, the artificial antigen and the antibody thereof have simple preparation method and low cost, and have wide application prospect in detecting phenylbutazone.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to phenylbutazone hapten, artificial antigen and antibody as well as preparation methods and application thereof.
Background
Phenylbutazone (phenylbutazone) is a antipyretic analgesic anti-inflammatory drug acting on the central nervous system, and is mainly used for treating rheumatic arthritis, rheumatoid arthritis, ankylosing spondylitis and the like. The long-term administration can cause adverse reactions such as nausea, vomiting, gastrointestinal discomfort, peptic ulcer, gastrointestinal bleeding and the like. Because of the strong anti-inflammatory and analgesic effects, the Chinese herbal medicine tea is often added into self-made herbal tea by a plurality of illegal merchants to blow the Chinese herbal medicine tea, and the health and market order of consumers are greatly jeopardized.
The currently known detection methods of phenylbutazone mainly comprise a liquid chromatography-ultraviolet detection method (GB/T20754-2006, method for measuring the residual quantity of phenylbutazone in livestock and poultry meat, liquid chromatography-ultraviolet detection method), an ultra-high performance liquid chromatography-tandem mass spectrometry (Xiong Shuang and the like, research for simultaneously measuring phenylbutazone and phenylbutazone in acne-removing cosmetics by using the ultra-high performance liquid chromatography-tandem mass spectrometry, analytical test school report, 2017, volume 36, 8) and the like. The method has the advantages of high detection efficiency, large detection amount, high accuracy and the like, but the detection equipment is expensive, the pretreatment process of the sample is complicated, professional operation is needed, the method is not suitable for the field detection requirement of a large number of samples, and most of detection objects are meat products and cosmetics. At present, no report related to the detection of phenylbutazone by an immune method exists, but the immune detection method is used as a high-efficiency, rapid, simple and convenient rapid detection method, and has the advantages of high sensitivity, strong specificity, convenient detection and the like. Therefore, the establishment of an immune detection method aiming at the illegal addition of phenylbutazone in herbal tea is particularly urgent. The key point of the establishment of the immune detection method is to design a proper phenylbutazone artificial antigen and obtain an antibody with high sensitivity and strong specificity, but no related report on the phenylbutazone artificial antigen, the antibody and the like is available at present.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the prior art and provide phenylbutazone hapten.
The invention also aims to provide a preparation method of the phenylbutazone hapten.
It is a further object of the present invention to provide the use of phenylbutazone hapten.
It is still another object of the present invention to provide a phenylbutazone artificial antigen.
It is a further object of the present invention to provide the use of the phenylbutazone artificial antigen.
It is still another object of the present invention to provide a phenylbutazone antibody.
It is still another object of the present invention to provide a kit for detecting phenylbutazone.
It is still another object of the present invention to provide an immunoassay for detecting phenylbutazone.
The above object of the present invention is achieved by the following technical solutions:
a phenylbutazone hapten BTS, wherein the structural formula of the phenylbutazone hapten is shown as a formula (I):
the phenylbutazone hapten BTS is named as follows by adopting a system naming method: 6- (3, 5-dioxo-1, 2-diphenylpyrazolidin-4-yl) hexanoic acid, i.e., 4-hexanoic acid-1, 2-diphenyl-3, 5-pyrazolidinedione.
The preparation method of phenylbutazone hapten BTS comprises the steps of reacting 1, 2-diphenyl-3, 5-pyrazolidinedione with 6-bromohexanoic acid ethyl ester in an alkaline environment, separating and purifying reactants, and hydrolyzing in the alkaline environment to obtain phenylbutazone hapten shown in a formula (I).
As a preferred embodiment, the preparation method of phenylbutazone hapten BTS comprises: fully dissolving 1, 2-diphenyl-3, 5-pyrrolidone in acetonitrile, adding K 2 CO 3 The solution was provided with an alkaline environment, naI was added as catalyst, followed by ethyl 6-bromohexanoate. Firstly, reacting in an ice water bath at 0 ℃ for 10min, removing air filled nitrogen into the whole reaction system, condensing and refluxing at 40-55 ℃ for 8-22 h, extracting with ultrapure water and ethyl acetate at 1:1, evaporating and collecting an obtained organic phase by a rotary evaporator, dissolving with methanol, adding ultrapure water with the methanol ratio of 1:2, stirring with 1mol/L sodium hydroxide aqueous solution at room temperature for 3-5 h, and adjusting pH to 6-7 by hydrochloric acid with the concentration of 1mol/L after the reaction is finished, thus obtaining the phenylbutazone hapten BTS shown in the formula (I).
Preferably, the molar ratio of the 1, 2-diphenyl-3, 5-pyrrolidone to the ethyl 6-bromohexanoate is 1:2.
Preferably, the temperature of the condensed reflux is 50 ℃ and the time is 12 hours.
Preferably, the time of stirring with sodium hydroxide solution at room temperature is 4h.
The invention also provides application of the phenylbutazone hapten in preparation of phenylbutazone artificial antigen.
A phenylbutazone artificial antigen, the structural formula of which is shown as a formula (II):
wherein P is a carrier protein.
Preferably, the carrier protein is selected from bovine serum albumin (Bovine serum albumin, BSA) or keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH).
The preparation method of the phenylbutazone artificial antigen is that carrier protein is coupled on carboxyl of phenylbutazone hapten BTS shown in a formula (I) through an active ester method.
The invention also provides application of the phenylbutazone artificial antigen in preparation of phenylbutazone antibodies.
A phenylbutazone antibody is prepared by immunizing animals with any one of the phenylbutazone artificial antigens.
Preferably, the phenylbutazone antibody is a polyclonal antibody.
Preferably, the phenylbutazone antibody is prepared by immunizing New Zealand white rabbits with phenylbutazone artificial antigen (BTS-KLH) taking carrier protein as key hole hemocyanin (KLH).
The application of the phenylbutazone antibody in phenylbutazone detection and/or phenylbutazone detection kit preparation is also within the protection scope of the invention.
A kit for detecting phenylbutazone comprises the phenylbutazone artificial antigen and an antibody prepared by immunizing animals with the phenylbutazone artificial antigen.
Preferably, the kit comprises antibodies prepared by immunizing animals with phenylbutazone artificial antigen (BTS-BSA) taking carrier protein as Bovine Serum Albumin (BSA) as a coating antigen and phenylbutazone artificial antigen (BTS-KLH) taking carrier protein as Keyhole Limpet Hemocyanin (KLH).
Preferably, the kit further comprises one or more of an enzyme label plate, phenylbutazone standard, enzyme conjugate, chromogenic solution, stop solution or wash solution.
An immunoassay method for detecting phenylbutazone, which uses the phenylbutazone artificial antigen as a coating source and uses the phenylbutazone antibody as a detection antibody for detection; the immunoassay method is a method of non-diagnostic therapeutic interest.
Preferably, the antibody prepared by immunizing an animal with phenylbutazone artificial antigen (BTS-BSA) taking carrier protein as Bovine Serum Albumin (BSA) as a coating antigen and phenylbutazone artificial antigen (BTS-KLH) taking carrier protein as Keyhole Limpet Hemocyanin (KLH) as an immunogen is used as a detection antibody for detection.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
A phenylbutazone colloidal gold rapid detection test strip comprises a base plate, and a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged on the base plate, wherein a invisible detection line and an invisible quality control line are printed on the nitrocellulose membrane, the invisible detection line is printed by adopting a coating antigen solution, and the invisible quality control line is printed by adopting a goat anti-rabbit antibody; the coating antigen is any of the phenylbutazone artificial antigens described above.
Preferably, the coating antigen is phenylbutazone artificial antigen (BTS-BSA) in which the carrier protein is Bovine Serum Albumin (BSA).
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a phenylbutazone hapten, and an artificial antigen and an antibody for detecting phenylbutazone are prepared by using the hapten, the antibody has high sensitivity and high specificity recognition capability on phenylbutazone, the half inhibition concentration of phenylbutazone is 3.40ng/mL, the detection range is 0.21-53.64 ng/mL, the minimum detection limit is 0.04ng/mL, and no cross reaction is caused on other structural and functional analogues such as piroxicam, ibuprofen, aminopyrine, indomethacin, and the like, so that the prepared phenylbutazone antibody has high detection sensitivity and strong specificity on phenylbutazone, can meet the requirements on site detection of a large number of samples, and an immunoassay method of phenylbutazone with higher specificity and sensitivity is established; in addition, the preparation method of the phenylbutazone hapten, the artificial antigen and the antibody thereof is simple and cost-effective, and has wide application prospect in phenylbutazone detection.
Drawings
FIG. 1 is a synthetic route diagram of hapten BTS of the present invention.
Fig. 2 is a graph of ultraviolet full wave scanning authentication results for BTS, BTS-KLH, KLH.
FIG. 3 is a graph showing the identification results of the ultraviolet full-wave scanning of BTS, BTS-BSA and BSA.
FIG. 4 is a graph of an indirect competition ELISA standard for detecting phenylbutazone.
Fig. 5 is a schematic side structural diagram of phenylbutazone colloidal gold immunochromatographic test strip, in which 1: a PVC bottom plate; 2: a sample pad; 3: a bonding pad; 4: NC film; 5: a detection line (T); 6: a quality control line (C); 7: a water absorbing pad.
FIG. 6 is a graph showing the judgment of the result of the phenylbutazone colloidal gold immunochromatography test strip.
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 of phenylbutazone hapten BTS
1. Preparation of phenylbutazone hapten BTS
200mg of 1, 2-diphenyl-3, 5-pyrrolidone was sufficiently dissolved in acetonitrile, and 250mg of K was added 2 CO 3 The solution was provided with an alkaline environment, 120mg of NaI was added as catalyst, and 150. Mu.L of ethyl 6-bromohexanoate was added. Firstly, reacting for 10min in an ice-water bath at the temperature of 0 ℃, then removing air-filled nitrogen into the whole reaction system, and condensing at the temperature of 40-55 DEG CReflux reaction for 8-22 h, extracting with ultrapure water and ethyl acetate at a ratio of 1:1, evaporating and collecting the obtained organic phase by a rotary evaporator, dissolving by methanol, adding ultrapure water with a ratio of 1:2 to methanol, stirring and reacting with 1mol/L sodium hydroxide aqueous solution at room temperature for 3-5 h, and regulating pH to 6-7 by hydrochloric acid with a concentration of 1mol/L after the reaction is finished, thereby obtaining hapten BTS, wherein a synthetic route diagram is shown in figure 1.
2. Identification of phenylbutazone hapten BTS
And (3) carrying out mass spectrum identification and nuclear magnetic resonance hydrogen spectrum identification on the phenylbutazone hapten BTS prepared in the step (1).
The hydrogen spectrum nuclear magnetic resonance results are as follows: 1H NMR (600 MHz, methanol-d 4) delta 7.41-7.32 (m, 6H), 5.34 (s, 0H), 4.86 (s, 124H), 4.29 (s, 0H), 3.31 (p, J=1.6 Hz, 19H), 2.56 (s, 1H), 2.49-1.99 (m, 2H), 2.35-1.18 (m, 10H), 1.33 (s, 13H), 1.30 (s, 7H).
The mass spectrum results were as follows: MS: c (C) 21 H 22 N 2 O 4 ：366.16，ESI - [M-H] - ：365.16
As can be seen from the mass spectrum nuclear magnetism result, 365.16 is the negative ion molecular peak of hapten 4- ((1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) (methyl) amino) butyric acid, the relative molecular mass is calculated to be 366.2, and accords with the actual relative molecular mass, which indicates that phenylbutazone hapten BTS is successfully prepared, the structure of which is shown as formula (I) and is named as: 4-hexanoic acid-1, 2-diphenyl-3, 5-pyrazolidinedione.
Example 2 preparation and identification of phenylbutazone Artificial antigen
1. Synthesis of artificial antigen
Phenylbutazone hapten BTS prepared in example 1 is respectively coupled with keyhole limpet hemocyanin KLH (keyhole limpet hemocyanin) and bovine serum albumin BSA (Bovine serum albumin) by an active ester method. The method specifically comprises the following steps:
hapten is respectively dissolved in 200 mu L of DMF, 1.5eq of carbodiimide (EDC) and 1.5eq of N-hydroxysuccinimide (NHS) are respectively added into the solution, and the solution is reacted for 4 hours at 4 ℃ and is marked as A solution; the carrier protein was dissolved in PBS buffer to complete dissolution, designated as solution B. Slowly adding the solution A into the solution B dropwise under magnetic stirring, and reacting for 12h at 4 ℃; and (3) putting the reaction solution into a dialysis bag, dialyzing in a phosphate buffer solution for 3 days at the temperature of 4 ℃, replacing the dialyzate three times a day, collecting the solution in the dialysis bag to obtain phenylbutazone artificial antigen-KLH, -BSA, and subpackaging at the temperature of-20 ℃ for preservation.
2. Identification of artificial antigens
The KLH, BSA, hapten BTS, artificial antigen BTS-KLH and BTS-BSA were identified by ultraviolet full wavelength method (200-400 nm).
The ultraviolet full-wavelength scanning identification results are shown in figures 2 and 3, and the highest absorbance of KLH, BSA, hapten BTS, artificial antigen BTS-KLH and BTS-BSA before and after coupling is compared, so that obvious differences exist between the absorption curves of BTS-KLH and BTS-BSA and the absorption curves of BTS-KLH and BTS-BSA, and the deviation different from the absorption curves of KLH and BSA appears at 280 nm; therefore, the absorption curves of BTS-KLH and BTS-BSA are the accumulated absorption peaks of KLH and BSA and hapten BTS respectively; the successful coupling of hapten BTS, protein KLH and BSA is shown, namely, artificial antigens BTS-KLH and BTS-BSA are successfully prepared.
Example 3 preparation of antibodies for detection of phenylbutazone
(1) Taking two healthy 6-week New Zealand white rabbits (one each for female and male), performing subcutaneous multipoint injection on the back after primary immunization is emulsified by Freund's complete adjuvant, wherein each immunization dose is 500 mug of artificial antigen coupled with keyhole limpet hemocyanin; the immunization was then boosted every three weeks, emulsified with Freund's incomplete adjuvant, four times in total.
(2) And (3) taking blood from the rabbit ear vein every week after the third immunization, centrifuging to obtain supernatant, and preserving at-20 ℃ for ELISA detection of the immune effect.
(3) Taking blood from heart after the fifth immunization, incubating the obtained rabbit blood at 37 ℃ for 2 hours, standing overnight at 4 ℃ for 12 hours, taking out the clear liquid of the water layer the next day, centrifuging at 6000r/min for 15 minutes at 4 ℃, removing the precipitate, obtaining the supernatant, sub-packaging and marking, and preserving at-20 ℃ to obtain the antibody for detecting phenylbutazone.
Example 4 establishment of an Indirect competitive ELISA assay for phenylbutazone
1. Method of
(1) The artificial antigen BTS-BSA is used as a coating source, the coating liquid is diluted to 1 mug/mL, a 96-well ELISA plate is coated, 100 mug of the ELISA plate is added into each well, and the ELISA plate is incubated overnight at 37 ℃ for 12 hours;
(2) Removing the coating liquid, washing for 2 times, and beating to dry;
(3) Adding 120 μl of blocking solution (i.e. 1% fish gelatin protein) into each well, and blocking at 37deg.C for 30min;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, taking out, and bagging for standby by self-sealing;
(5) Diluting phenylbutazone antibody with PBST 1:16000 times, and diluting phenylbutazone drug to 1000ng/mL,100ng/mL,10ng/mL,1ng/mL,0.1ng/mL,0.01ng/mL,0.001ng/mL;
(6) 50. Mu.L phenylbutazone diluent (four groups are in parallel) is added to each row, 50. Mu.L/Kong Tu serum diluent is added, incubation is carried out for 40min at 37 ℃, and washing is carried out for 5 times;
(7) Adding goat anti-rabbit secondary antibody-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and drying;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 10% H was added 2 SO 4 The reaction was terminated and the OD was read at 450 nm.
2. Experimental results
As shown in FIG. 4, the standard curve of the indirect competition ELISA for detecting phenylbutazone, the half Inhibitory Concentration (IC) of the antibody for detecting phenylbutazone was found 50 ) 3.40ng/mL, the quantitative detection range is 0.21-53.64 ng/mL, and the lowest detection limit is 0.04ng/mL; the antibody for detecting phenylbutazone prepared by the method can meet the detection requirement, and has high identification capability, high specificity and high detection sensitivity for phenylbutazone.
Example 5 evaluation of specificity of antibodies for detecting phenylbutazone
1. Test method
The specificity for detecting phenylbutazone was determined by conducting a cross-reaction experiment with phenylbutazone and its analogues, the specificity of the antibodies being expressed as cross-reactivity (CR), the smaller the cross-reactivity the stronger the specificity. Phenylbutazone and analogues thereof (piroxicam, ibuprofen, aminopyrine, indomethacin) were diluted in multiple ratios and measured by indirect competition ELISA, and IC of each analogue was obtained by the same sensitivity verification method as in example 5 50 Values, phenylbutazone cross-reactivity (CR) was calculated according to the following formula:
CR(％)＝IC 50 (phenylbutazone)/IC 50 (analog) ×100%.
2. Experimental results
The results of the cross-reaction of phenylbutazone and its analogues are shown in Table 1, and it is found that the antibody for detecting phenylbutazone has a cross-reaction rate of 100% with phenylbutazone and IC 50 The cross reaction rate of the piroxicam, ibuprofen, aminopyrine and indomethacin is 3.40ng/mL and is less than 1%; the antibody for detecting phenylbutazone has strong specificity, can effectively eliminate the interference of the analogue to phenylbutazone detection, and can be specially used for detecting phenylbutazone.
TABLE 1 Cross-reaction results of phenylbutazone and its analogues
EXAMPLE 6 development of ELISA kit for detecting phenylbutazone
1. A kit for detecting phenylbutazone is constructed, the kit comprising the following parts:
(1) Preparing an ELISA plate coated with a coating source: diluting the antigen BTS-BSA prepared in example 3 as a coating source to 125ng/mL by using a coating buffer solution, adding 100 mu L of the coating source into each hole, incubating overnight at 37 ℃ in a dark place, pouring out the liquid in the holes, washing 2 times by using a washing solution for 30 seconds each time, beating dry, adding 120 mu L of a sealing solution into each hole, incubating for 3 hours at 37 ℃ in a dark place, pouring out the liquid in the holes, beating dry, and vacuum sealing and storing by using an aluminum film after 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) Phenylbutazone standard solution: 7 concentration gradients of 1000ng/mL,100ng/mL,10ng/mL,1ng/mL,0.1ng/mL,0.01ng/mL,0.001ng/mL, respectively;
(3) Phenylbutazone polyclonal antibody prepared in example 4;
(4) Enzyme conjugate: the phenylbutazone clone antibody prepared in example 4 marked by horseradish peroxidase;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) H with 10% stop solution 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 (ng/mL) of the phenylbutazone 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 phenylbutazone in the sample.
Example 7 development of phenylbutazone colloidal gold immunochromatographic test strip
1. Preparation of gold-labeled antibodies and gold-labeled conjugate pads
The colloid Jin Xuanfu liquid with the average diameter of 40nm is prepared by adopting a method of reducing chloroauric acid by trisodium citrate.
The colloidal gold is firstly treated with 0.2mol of K 2 CO 3 The pH of the solution was adjusted to 8.5, then the amount of antibody labeling was determined by classical NaCl titration, and finally 20. Mu.g of phenylbutazone antibody prepared in example 3 and 1mL of colloidal gold solution were selected for labeling to obtain gold-labeled antibody, which was stored at 4 ℃. Spraying 4% BSA solution on glass wool with an XYZ-3000 three-dimensional film spraying instrument at a volume of 8 mu L/cm, drying at a temperature of 42 ℃ for 50min by using a drying oven, spraying gold-labeled antibody on the glass wool with a volume of 6 mu L/cm, drying at a temperature of 42 ℃ for 50min by using a drying oven, and vacuum drying for preservation.
2. Conjugated antigen sheep anti-rabbit coated cellulose membrane
Coating antigen with the concentration of 1mg/mL is sprayed on the lower side of the cellulose membrane in the amount of 1.2 mu L/cm by using an XYZ-3000 three-dimensional film spraying instrument to serve as a detection line. Sheep anti-rabbit IgG with a concentration of 120. Mu.g/L was sprayed on the upper side of the cellulose membrane in an amount of 1.2. Mu.L/cm using an XYZ-3000 three-dimensional film spraying apparatus as a control line, with the two lines being 8mm apart.
3. Assembling of quick test paper strip
As shown in fig. 5, the cellulose film 4 was stuck to the middle portion of the backing plate 1, and the water absorbing pad 7 was stuck to the cellulose film 4, and the upper side and the cellulose film 4 were overlapped by 1mm. The gold-labeled conjugate pad 3 was adhered under the cellulose membrane 4 to overlap by 1mm. The sample pad 2 is adhered under the gold-labeled conjugate pad 3 to overlap by 2mm. The assembled test paper board was cut into test strips 3.05mm wide with a chopper.
5. Extraction and preparation of herbal tea sample
Sucking 0.5mL of herbal tea, adding 9.5mL 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.
6. Quick detection test strip detection and judgment
After the sample solution to be tested is added into the test paper strip or the test paper card test end, the sample solution to be tested drives the sample to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse towards the cellulose membrane 4 together through the siphon action, and finally permeates into the end of the water absorption pad 7. In the diffusion process, if an object to be detected exists in a sample, the object to be detected is combined with the gold-labeled antibody, so that an antigen binding point on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and a invisible detection line 5 (a conjugate of hapten and carrier protein) on a cellulose membrane 4 is prevented, and the invisible detection line 5 is not developed or developed very weakly, namely, positive or weak positive of the detection sample is indicated; if the sample to be detected is not contained in the sample, the gold-labeled antibody shows a clear red line when encountering the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the gold-labeled antibody is also combined with the invisible quality control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible quality control line 6 is red. The presence or absence of the color of the invisible quality control line 6 indicates the validity or invalidity of the test strip, and the determination result is shown in fig. 6.
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 (7)
1. The phenylbutazone hapten is characterized in that the structural formula is shown as a formula (I):
formula (I).
2. The preparation method of phenylbutazone hapten as claimed in claim 1, characterized in that 1, 2-diphenyl-3, 5-pyrazolidinedione and 6-bromohexanoic acid ethyl ester are reacted in alkaline environment, the reactants are separated and purified, and then hydrolyzed in alkaline environment, so as to obtain phenylbutazone hapten shown in formula (I).
3. Use of phenylbutazone hapten as claimed in claim 1 for the preparation of phenylbutazone artificial antigen.
4. The phenylbutazone artificial antigen is characterized in that the structural formula is shown as a formula (II):
(II)
Wherein P is a carrier protein.
5. The phenylbutazone artificial antigen of claim 4, wherein the carrier protein is selected from bovine serum albumin or keyhole limpet hemocyanin.
6. The method for preparing phenylbutazone artificial antigen according to claim 4 or 5, wherein the carrier protein is coupled to the carboxyl group of phenylbutazone antigen according to claim 1 by an active ester method.
7. Use of the phenylbutazone artificial antigen according to claim 4 or 5 for the preparation of phenylbutazone antibodies.
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