CN211453649U - Immunochromatography test strip based on quantum dot fluorescent microspheres - Google Patents
Immunochromatography test strip based on quantum dot fluorescent microspheres Download PDFInfo
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Abstract
The utility model discloses an immunochromatography test strip based on quantum dot fluorescent microspheres, which comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is lapped on the upper surface of one end of the nitrocellulose membrane, the water absorption pad is connected on the upper surface of the other end of the nitrocellulose membrane, and a detection line and a quality control line which are parallel to the end surface of the bottom plate are coated on the exposed nitrocellulose membrane between the end part of the sample pad and the end part of the water absorption pad; 1mg/mL cyproheptadine hydrochloride competitive antigen is fixed on the surface of the detection line; goat anti-mouse IgG with the concentration of 0.3mg/mL is fixed on the surface of the quality control line; the test strip can realize rapid qualitative and quantitative detection of cyproheptadine hydrochloride by a fluorescent card reader, and the minimum detection limit reaches 0.096 ng/mL; the test strip provided by the utility model has the characteristics of easy operation, fast detection speed, high sensitivity and the like, and can be used for the residual determination of cyproheptadine hydrochloride in pig urine.
Description
Technical Field
The utility model belongs to the technical field of immunoassay, concretely relates to quantum dot fluorescence microsphere immunochromatography test paper strip of short-term test cyproheptadine hydrochloride.
Background
Cyproheptadine hydrochloride is used as a medical drug for treating various allergic diseases such as skin itch, contact dermatitis, allergic rhinitis and the like, and has the effects of inhibiting the satiety center of the hypothalamus, stimulating appetite, increasing body weight and promoting growth. The cyproheptadine hydrochloride is illegally added into animal feed, can generate similar effect with clenbuterol hydrochloride, and can promote the growth of livestock and poultry and improve the lean meat percentage, so the cyproheptadine hydrochloride is called as novel clenbuterol. If people eat meat food with cyproheptadine hydrochloride residue for a long time, the health of human bodies can be seriously damaged, adverse symptoms such as hypodynamia, dizziness, sleepiness, respiratory depression and the like are generated, and serious people can cause coma and even death, and the meat food is particularly sensitive to children and old people. Therefore, the chinese department of agriculture issued 1519 bulletin in 2010, strictly prohibiting the use of cyproheptadine hydrochloride in feed and animal drinking water or the addition of cyproheptadine hydrochloride.
At present, the detection method of cyproheptadine hydrochloride mainly adopts instrument detection, such as a high performance liquid chromatography and a liquid chromatography-mass spectrometry detection method. These detection methods require a lot of time for detection, require expensive detection equipment, require special experimenters for operation, and are not favorable for rapid detection of a large number of samples. The enzyme-linked immunoassay (ELISA) method has the advantages of high sensitivity, good recovery rate, strong specificity and the like, can realize on-site rapid qualitative and semi-quantitative analysis, and is widely applied to drug residue detection, but the method still needs professional training of operators. The immunochromatographic test strip based on the ELISA principle does not need professional training operators, has the characteristics of simplicity and rapidness, is lower in cost, high in sensitivity and free of complex instruments and equipment, and is widely applied to the fields of biological medicines, pesticide and veterinary drug residues, food safety detection and the like.
The quantum dot fluorescent microsphere immunochromatography is an immunochromatography technology established by taking quantum dot fluorescent microspheres as markers. The quantum dots have good biocompatibility, and can be specifically coupled with biomolecules such as nucleic acid, antibodies, DNA and the like after the surfaces of the quantum dots are chemically modified. The quantum dot fluorescent microsphere is a fluorescent marker formed by embedding quantum dot fluorescent dye or adsorbing the quantum dot fluorescent dye on a high polymer carrier, can effectively improve the reaction sensitivity, is regarded as the fluorescent marker with the most development potential in the technical field of immunochromatography detection, the process of coupling the quantum dot fluorescent microsphere and an antibody is a key part of the whole method, and the coupling effect finally influences the improvement of the sensitivity. At present, the quantum dot technology is mainly used for detecting protein biomarkers and other small molecule compounds. The detection of cyproheptadine hydrochloride by using quantum dot fluorescent microsphere immunochromatography has not been reported yet.
SUMMERY OF THE UTILITY MODEL
The utility model provides an immunochromatography test strip based on quantum dot fluorescent microspheres, which can realize simple, convenient, rapid and highly sensitive quantitative detection of cyproheptadine hydrochloride. The utility model is realized by the following technical scheme:
firstly, the utility model provides an immunochromatography test strip based on quantum dot fluorescent microspheres, which comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is lapped on the upper surface of one end of the nitrocellulose membrane, the water absorption pad is connected on the upper surface of the other end of the nitrocellulose membrane, and a detection line and a quality control line which are parallel to the end surface of the bottom plate are coated on the exposed nitrocellulose membrane between the end part of the sample pad and the end part of the water absorption pad;
1mg/mL cyproheptadine hydrochloride competitive antigen is fixed on the surface of the detection line;
goat anti-mouse IgG with the concentration of 0.3mg/mL is fixed on the surface of the quality control line;
in specific implementation, before the sample pad is assembled, the sample pad is preferably soaked in a buffer solution for 24 hours, and the buffer solution comprises the following components: calculated by mass percentage, 2 percent BSA, 2.5 percent cane sugar and 0.02 percent sodium azide NaN3The balance is made up with PBS, pH 7.4;
the detection sensitivity of the test strip to cyproheptadine hydrochloride reaches 0.096 ng/mL.
Further, the utility model provides an among the immunochromatography test paper strip based on quantum dot fluorescence microballon, the interval between detection line and the matter accuse line is 3-8 mm.
Secondly, the utility model also provides an above-mentioned immunochromatography test strip based on quantum dot fluorescence microballon is in the application of detecting cyproheptadine hydrochloride. The specific detection method comprises the following steps:
(1) respectively mixing the anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe with a cyproheptadine hydrochloride standard substance, incubating for 1min, dripping the mixture on a sample pad, after 15min, recording the fluorescence intensity of a test strip detection line (T line) and a quality control line (C line) by using a fluorescence reading instrument if the quality control line of the test strip is colored under the irradiation of an ultraviolet lamp, establishing an inhibition rate-cyproheptadine hydrochloride standard substance concentration linear relation through calculation, and drawing a standard curve: y =95.81-99.22/(1+ (x/0.9)0.83) (ii) a If the quality control line (C line) of the test strip does not develop color under the irradiation of the ultraviolet lamp, the test strip is judged to be invalid; the concentration of the cyproheptadine hydrochloride standard substance is 0-50 ng/mL (figure 5), and the preferable concentration of the cyproheptadine hydrochloride standard substance is 0, 0.78, 1.56, 3.125, 6.25,12.5, 25 and 50 ng/mL (figure 4) in sequence.
(2) Placing a sample to be tested in a centrifuge tube, and centrifuging for 10 min at 3000 g; sucking the supernatant, mixing the supernatant with a diluent according to the volume ratio of 1:4 to obtain a sample diluent, and repeating the step (1) to detect: if the quality control line of the test strip is colored under the irradiation of an ultraviolet lamp, the content of the cyproheptadine hydrochloride can be judged according to the color development degree of the detection line, the fluorescence intensity of the detection line and the quality control line of the test strip is recorded by a fluorescence reading instrument, the inhibition rate is calculated according to the fluorescence value and is substituted into the curve equation in the step (1), and the content of the cyproheptadine hydrochloride in the sample is obtained; if the quality control line does not develop color under the irradiation of ultraviolet rays, the test strip is judged to be invalid. Further, in the detection method, the anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent microsphere probe is obtained by the following method:
dissolving 0.12 mg of quantum dot fluorescent microspheres in 2.7 mL of 0.01 mol/L phosphate buffer solution with pH of 6.0, adding 1 mu g of EDC for activation, then adding 150 mu g of cyproheptadine hydrochloride monoclonal antibody, uniformly mixing, reacting for 30 min by magnetic stirring, centrifuging at 13500 rpm for 15min, removing supernatant, redissolving the centrifugal precipitate by 600 mu L of redissolution, and storing at 4 ℃.
The formula of the redissolution is as follows: calculated by mass percentage, 5 percent of sucrose, 2 percent of fructose, 1 percent of PEG 20000, 1 percent of BSA, 0.4 percent of Tween-20 and deionized water are used for making up the balance.
The method for preparing the anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe in the step can also be seen in the literature: "RenM, Xu H, Huang X, et al, immunochlorogenic assay for ultrasensitive detection of afflatoxin B1in maize by highly luminescent quantum dot beads.[J]. Acs Applied Materials&Interfaces, 2014, 6(16):14215-14222.”。
The utility model discloses in, quantum dot fluorescence microballon is preferred to be obtained through doping CdSe/ZnS quantum dot, and the diameter is 247nm 13 nm, and this quantum dot fluorescence microballon is prior art, but the market purchase or prepare by oneself, and the preparation method is seen the literature: "Ren M, Xu H, Huang X, et al, immunochlorogenic assay for ultrasensitive detection of aflatoxin B1in maize by highly luminescentquantum dot beads.[J]. Acs Applied Materials&Interfaces, 2014, 6(16):14215-14222.”。
Compared with the prior detection method, the utility model provides a test strip and detection beneficial effect thereof:
(1) the utility model provides a pair of hydrochloric acid cyproheptadine immunochromatography test paper strip based on quantum dot fluorescence microballon, more traditional analytical technique can realize quicker, simple and convenient, the sensitive detection to hydrochloric acid cyproheptadine, utilizes portable fluorescence card reader can carry out on-the-spot real-time quantitative determination. The test strip can be used for detecting cyproheptadine hydrochloride in various sample sources, such as pig urine, animal feed, pork tissue and the like.
(2) The utility model provides a pair of hydrochloric acid cyproheptadine fluorescence immunochromatography test paper strip based on quantum dot fluorescence microballon owing to introduced quantum dot fluorescence probe, has improved the detectivity of this method greatly, and its minimum detection limit is better than the ELISA method with the antibody.
(3) The utility model discloses a detection method of hydrochloric acid cyproheptadine immunochromatography test paper strip based on quantum dot fluorescence microballon can realize the quantitative determination to hydrochloric acid cyproheptadine in the sample.
Drawings
FIG. 1 is a standard inhibition curve of monoclonal antibody to cyproheptadine hydrochloride by indirect competitive ELISA.
FIG. 2 is a schematic diagram of the principle of the cyproheptadine hydrochloride immunochromatographic test strip based on quantum dot fluorescent microspheres of the utility model
FIG. 3 is a schematic diagram showing the interpretation of the results of the rapid detection of cyproheptadine hydrochloride of the present invention.
FIG. 4 is a graph showing the sensitivity test for detecting cyproheptadine hydrochloride at different concentrations.
Fig. 5 is a standard curve of the immunochromatographic test strip for detecting cyproheptadine hydrochloride of the present invention.
Detailed Description
EXAMPLE 1 preparation of cyproheptadine hydrochloride monoclonal antibody
1. Synthesis of immunogen (CYP-BSA), Competition antigen (CYP-OVA)
1.1, selecting 0.05 mmol of cyproheptadine hapten hydrochloride, dissolving the cyproheptadine hapten in 1 mL of Dimethylformamide (DMF), sequentially adding N-hydroxysuccinimide (NHS) (0.06 mmol) and N, N-Dicyclohexylcarbodiimide (DCC) (0.06 mmol), stirring at normal temperature in a dark environment, and standing overnight.
1.2 day 2, the mixture left overnight was centrifuged at 10000 g for 10 min and the supernatant was collected for further use.
1.3 BSA (0.15 × 10)-3mmol) and OVA (0.15 × 10)-3mmol) are respectively dissolved in 5 mL PBS to prepare protein solution; slowly dripping the solution obtained in the step 1.2 into a protein solution, and stirring for 4 hours at normal temperature;
1.4 after completion of the reaction, the product was placed in 4oDialyzing in PBS for three days, changing dialysate for 3-4 times per day, dialyzing to obtain immunogen (CYP-BSA) and competitive antigen (CYP-OVA), and placing in-20oAnd C, storing under the environment.
In the step, the cyproheptadine hapten hydrochloride can be purchased commercially or prepared by self, and the preparation method is a conventional technology in the field, for example, see a preparation method disclosed in the research of a ' Yanrubiru ' novel ' clenbuterol hydrochloride immunoassay technology [ D ].
2. Identification of artificial antigen of cyproheptadine hydrochloride
The synthesized cyproheptadine hydrochloride immune antigen, the competitive antigen and the carrier protein are respectively scanned by ultraviolet light at 200-400 nm, and the combination ratio of the synthesized cyproheptadine hydrochloride immune antigen, the competitive antigen and the carrier protein is calculated by comparing the light absorption values of the synthesized cyproheptadine hydrochloride immune antigen, the competitive antigen and the carrier protein under the same wavelength.
The ultraviolet spectrogram of the immunogen and the competitive antigen shifts compared with the maximum absorption wavelength of the carrier protein, which indicates that the hapten and the carrier protein are successfully coupled to prepare the cyproheptadine hydrochloride artificial antigen. Through calculation, the binding ratio of the obtained artificial antigen cyproheptadine hapten molecule and the BSA molecule is 20:1, and the binding ratio of the cyproheptadine hapten molecule and the OVA molecule is 18:1, so that the coupling effect is better.
3. Preparation of cyproheptadine hydrochloride monoclonal antibody
Balb/c female mice 6-8 weeks old are selected and immunized four times, the dosage of each immunogen (CYP-BSA obtained in step 1.4) is 100 mu g/mouse, and the interval time of each immunization is 3 weeks. The primary immunization is carried out by mixing immune antigen and Freund's complete adjuvant in equal volume, and injecting into abdominal cavity. 150 mu g of immunogen is injected into each abdominal cavity 3 days before fusion for boosting, and blood is taken after tail breaking 6 days after immunization, and the titer is determined by adopting an ELISA method. Taking immune mouse spleen cells with the highest titer to mix with mouse myeloma cells SP2/0, carrying out cell fusion by using a fusion agent of 50% PEG, and screening hybridoma cells by adopting a step screening method when the cells grow to 1/4 of the area of a culture hole. Performing antibody property identification on cell supernatant by indirect ELISA method, and determining OD after cyproheptadine hydrochloride blocking450The nm value is reduced to below 50% of the control wells, the wells are positive, and the wells which are positive after 2-3 detections are immediately subcloned by a limiting dilution method. Performing expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernate, measuring titer by indirect ELISA, and freezing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, and injecting hybridoma into the abdominal cavity 7-10 days laterCell 1-2 × 106Ascites of the mice are extracted 7 to 10 days later, the supernatant is centrifuged, and the supernatant is frozen for later use after titer is determined.
The preparation methods of the steps 2 and 3 can also be referred to the methods disclosed in the literatures of "study on the immunologic detection technology of salicylaldehyde shavings, novel" clenbuterol "cyproheptadine hydrochloride [ D ]" and "preparation of general organophosphorus and camptothecin monoclonal antibodies and the immunologic detection technology study [ D ]".
4. Purification of ascites
4.1 mix 5 mL ascites with an equal volume of PBS, add 5 mL saturated ammonium sulfate solution to it, stir while adding dropwise, continue stirring at room temperature for 30 min, place in 4 ℃ refrigerator cooling overnight, make the immunoglobulin precipitate fully.
4.2 centrifuging the fully precipitated mixture, 4o10000 g/min, 20 min, dissolving the precipitate with 5 mL PBS, adding 3 mL saturated ammonium sulfate solution dropwise while stirring, stirring for 30 min, and standing for 4oAnd C, refrigerating the refrigerator overnight.
4.3 repeat step 4.2 twice, use 5 mL PBS to dissolve the last centrifugation sediment, put it into dialysis bag, use PBS dialysis for 3 days, change the liquid 3-4 times a day, fully remove the salt.
4.4 centrifugation of the dialyzed product 4oC, 5000 g/min, 50 min, supernatant fluid is cyproheptadine monoclonal antibody hydrochloride obtained by crude extraction, subpackaging and storing in-20oC, preparing for subsequent use.
The detection sensitivity of the monoclonal antibody is identified by adopting an indirect competitive ELISA method, and the detection method is shown in the literature' Yan reting preparation of general organophosphorus and camptothecin monoclonal antibodies and the research of immunoassay technology [ D]"the disclosed method. The detection result is shown in fig. 1, and a standard curve formula is drawn at the same time as: y =90.6-90.23/(1+ (x/6.52)0.95) Wherein x represents the concentration of the cyproheptadine hydrochloride standard substance, and y represents the inhibition rate of the cyproheptadine hydrochloride standard substance on the monoclonal antibody. As shown in FIG. 1, the cyproheptadine hydrochloride standard substance has a concentration (IC) of cyproheptadine hydrochloride corresponding to 50% of monoclonal antibody inhibition rate50) 8.05 ng/mL, lowest detection line (IC)10) 0.70 ng/mL, detection Range (IC)20-IC80) In the range of 1.69-54.46 ng/mL.
EXAMPLE 2 preparation of anti-cyproheptadine hydrochloride monoclonal antibody-Quantum dot fluorescent Probe
Dissolving 0.12 mg of quantum dot fluorescent microspheres in 2.7 mL of 0.01 mol/L phosphate buffer solution with pH of 6.0, adding 1 mu g of EDC for activation, then adding 150 mu g of cyproheptadine hydrochloride monoclonal antibody, uniformly mixing, reacting for 30 min by magnetic stirring, centrifuging at 13500 rpm for 15min, removing supernatant, redissolving the centrifugal precipitate by 600 mu L of redissolution, and storing at 4 ℃.
The formula of the redissolution is as follows: calculated by mass percentage, 5 percent of sucrose, 2 percent of fructose, 1 percent of PEG 20000, 1 percent of BSA, 0.4 percent of Tween-20 and deionized water are used for making up the balance.
The preparation method of the Quantum DoT fluorescent microspheres can also be found in the literature "Duan H, Chen X, Xu W, et. Quantum-DoT submicron-based immunochromatic fluorescent for obtaining and detecting the molecular structure [ J]Talanta, 2015, 132: 126-. The disclosed method is prepared; the above-mentioned method for preparing anti-cyproheptadine hydrochloride monoclonal antibody-quantum dot fluorescent probe can also be found in the literature "Ren M, Xu H, Huang X, et al1in maize by highly luminescentquantum dot beads.[J]. Acs Applied Materials&Interfaces, 2014, 6(16):14215-14222.”
Example 3 preparation of test strips
As shown in figure 2A, an immunochromatographic test strip based on quantum dot fluorescent microspheres comprises a PVC base plate, a sample pad 1, a nitrocellulose membrane 4 and a water absorption pad 5, wherein the sample pad 1 is lapped on one end upper surface (overlapped by 2 mm) of the nitrocellulose membrane 4, the water absorption pad 5 is connected on the other end upper surface (overlapped by 2 mm) of the nitrocellulose membrane 4, and a detection line 2 (T line) and a quality control line 3 (C line) parallel to the end face of the base plate are coated on the exposed nitrocellulose membrane 4 between the end part of the sample pad 1 and the end part of the water absorption pad 5.
As shown in fig. 2B, the assembled test strip is cut into small strips with a width of 4 mm and a length of 60mm by a strip cutter, and placed in a white strip-shaped flat test card, the surface of the test shell 6 is provided with a sample hole 7 and a test window 8, and a sample to be tested and a fluorescent probe are suitable for being injected onto the test strip from the sample hole 7; through inspection window 8, the operating personnel of being convenient for observe the experimental result.
1mg/mL cyproheptadine hydrochloride competitive antigen (CYP-OVA prepared in step 1.4 of example 1) is fixed on the surface of the detection line;
goat anti-mouse IgG with the concentration of 0.3mg/mL is fixed on the surface of the quality control line;
in this example, the sample pad was immersed in the buffer solution for 1 hour before assembly, and then was assembled after being dried at room temperature; the soaking step can reduce sample difference, improve experiment sensitivity, avoid the trouble of complex identifier and tracing buffer solution, and complete detection in one step.
The buffer solution comprises the following components: 2% BSA, 2.5% sucrose, 0.02% sodium azide NaN3, in mass percent, the balance being PBS, pH 7.4.
The goat anti-mouse IgG described above is commercially available, or the references "Liu B, Gong H, Wang Y, et al, agricultural immunochemical assay for biochemical analysis of pathological and enzymatic products in agricultural products [ J ]. ANALYTICAL METHODS, 2018, 10.
Example 4 application of test strip for detecting fluorescent microsphere of quantum dot
(1) Result judgment
Taking 1 mu L of the quantum dot fluorescent probe prepared in the embodiment 2, mixing the 1 mu L of the quantum dot fluorescent probe with 5% methanol PBS buffer solution and buffer solution dissolved with cyproheptadine hydrochloride standard products with different concentrations, incubating at room temperature for 1min, adding a test strip sample adding hole, reacting for 15min, and detecting by using a test strip reader. The experimental group without the addition of the cyproheptadine hydrochloride standard was used as a negative control group. After the test strip enters a reader (a dry fluorescence immunoassay analyzer, model: FIC-S2011-L44, manufactured by Suzhou and Mimeji precision instruments Co., Ltd.), the fluorescence signal is emitted by the quantum dot fluorescent microspheres at the T, C line of the test strip after being excited by an optical element, and the fluorescence signal is converted into a digital signal by the reader to be displayed on a display of the reader. After 15min, if the C line is developed and the color intensity of the T line is equivalent to that of the T line of the negative control group (as shown in FIG. 3B), the result is negative; if the T, C lines are all colored and the T line color intensity is smaller than that of the negative control group (as shown in FIG. 3C), or the C line is colored and the T line is not colored (as shown in FIG. 3D), the result is positive; if the line C does not develop color, the test strip is determined to be ineffective (see FIG. 3E, F), wherein FIG. 3A is a negative control. As shown in fig. 4, from left to right, the concentrations of the standard substances are respectively 0, 0.78, 1.56, 3.125, 6.25,12.5, 25 and 50 ng/mL, the brightness of the T line gradually becomes dark until the T line is invisible, the result is in line with the expected result of the experiment, and the qualitative detection of the cyproheptadine residue can be primarily realized.
(2) Determination of the Standard Curve
Diluting a cyproheptadine hydrochloride standard substance by using a PBS buffer solution containing 5% methanol by volume in a 2-fold gradient manner, wherein the concentrations are as follows: 0.048828125, 0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25,12.5, 25, 50, each concentration was measured in five replicates, after 15min the linear fluorescence intensity of test strip T, C was recorded using a fluorescence reader, and the FI for a 0 standard solution was setT/FICRatio B0Other FI at normalized concentrationT/FICThe ratio is B, in0-B）/B0× 100 (%) as ordinate and cyproheptadine hydrochloride concentration as abscissa, the test strip competition inhibition curve is plotted, the obtained standard curve is shown in FIG. 5, the formula of the inhibition curve is y =95.81-99.22/(1+ (x/0.9)0.83) And calculating according to a formula: IC (integrated circuit)50The detection line is 1.09 ng/mL, the lowest detection line is 0.096ng/mL, and the detection range is 0.22-6.68 ng/mL.
(3) Sample processing
Taking 2 mL of a pig urine sample, placing the pig urine sample in a polystyrene centrifuge tube, and centrifuging for 10 min at 3000 g; and (3) sucking the supernatant, and mixing with the diluent according to the ratio of 1:4, mixing by volume; an appropriate amount of the mixed solution (sample diluent) was taken for detection.
The diluent is PBS buffer solution containing 5% methanol by volume.
(4) Detection of samples
Adding a cyproheptadine hydrochloride standard substance into a urine sample without cyproheptadine hydrochloride to ensure that the final concentrations of the standard substance in the sample are respectively 1, 5 and 20 mg/L; and (3) respectively detecting the added samples according to the method, repeating each experiment for 5 times, and respectively calculating the variation coefficient and the addition recovery rate. The results are shown in Table 1, respectively.
TABLE 1 spiking recovery test results
| Additive concentration (mg/L) | Recovery rate | Coefficient of |
| 1 | 94% | 3.5% |
| 5 | 81% | 1.5% |
| 20 | 104% | 7% |
The result shows that the average adding recovery rate of the pig urine sample is 94-104%, and the coefficient of variation is 1.5-7%. The detection result shows that the method can accurately detect the content of cyproheptadine hydrochloride in pig urine.
(4) Cross reaction rate test
Selecting drugs with similar structure or function to cyproheptadine such as fletadine, ketotifen fumarate, and clonidine hydrochloride, performing cross reaction test, and obtaining IC of each drug according to standard curve50. Using IC' s50Cyproheptadine hydrochloride/IC50The analogs calculate the cross-reactivity of the test strip to other cyproheptadine analogs. The smaller the cross reaction rate with other drugs is, the smaller the cross reaction rate is, the smaller the molecular weight shows that the quantum dot fluorescent microsphere immunochromatographic test strip has on the hydrochloric acid cyproheptadineThe better the detection specificity of the pyridine. Result quantum dot fluorescent microsphere immunochromatographic test strip for cross-reacting rate of Frettdine, ketotifen and clonidine hydrochloride<1 percent, which shows that the method has better specificity, and the utility model can be used for detecting the residual quantity of cyproheptadine hydrochloride in the pig urine.
Claims (2)
1. An immunochromatography test strip based on quantum dot fluorescent microspheres comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorption pad, and is characterized in that the sample pad is lapped on the upper surface of one end of the nitrocellulose membrane, the water absorption pad is lapped on the upper surface of the other end of the nitrocellulose membrane, and a detection line and a quality control line which are parallel to the end face of the bottom plate are coated on the exposed nitrocellulose membrane between the end part of the sample pad and the end part of the water absorption pad; a cyproheptadine hydrochloride competitive antigen is fixed on the surface of the detection line; and goat anti-mouse IgG is fixed on the surface of the quality control line.
2. The immunochromatographic test strip based on quantum dot fluorescent microspheres of claim 1, wherein the interval between the detection line and the quality control line is 3-8 mm.
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