Immunochromatography test paper for detecting oseltamivir phosphate in eggs / meat and preparation method thereof

Abstract

The invention discloses immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat and a preparation method of the immunochromatographic test paper. The immunochromatographic test paper provided by the invention is a transverse flow immunochromatographic test strip detection method (LFIST) based on a competitive mode, and is used for rapidly and sensitively detecting oseltamivir phosphate (OP) residues in poultry products on site. The sensitivity (half inhibitory concentration, IC50) of the LFIST in detection of egg and chicken samples is confirmed to be 2.56 and 2.63 [mu] g / kg, and the limit of detection (LOD) value is 0.43 and 0.42 [mu] g / kg respectively; for intra-batch and inter-batch reproducibility, the recovery rate of an OP labeled sample is between 82.8% and 91.2%, and the coefficient of variation (CV) is less than 5.67% (intra-batch) and 6.52% (inter-batch); when an egg sample and a chicken sample are tested in parallel, the performance of the LFIST is equivalent to that of high performance liquid chromatography (HPLC); lFIST needs less than 5 minutes and does not depend on professionals, so that the LFIST can be used as a monitoring tool for on-site detection of OP residues.

Description

Technical Field

[0001] The invention belongs to the technical field of immunochromatographic detection, and particularly relates to an immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat and a preparation method thereof. Background Art

[0002] Oseltamivir phosphate (OP) is an ethyl ester raw drug widely used to treat and prevent influenza A and B virus infections. After oral administration of OP, OP is absorbed by the gastrointestinal tract and metabolized to oseltamivir carboxylic acid (OCA) by liver esterase. OCA is a potent and selective influenza virus inhibitor that binds to and inhibits the activity of the viral surface protein neuraminidase (NA). Specific inhibition of neuraminidase activity ultimately prevents the release of the virus from infected cells and alleviates symptoms associated with infection. OP residues may enter the human body through the food chain and affect human health. Studies have shown that exposure to excessive OP can cause gastrointestinal disorders, neuropsychiatric symptoms, and sudden death. Therefore, the detection of OP in agriculture and food is crucial to human health, and an OP residue monitoring system needs to be established.

[0003] To date, a variety of analytical methods for detecting OP have been reported, including high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), and liquid chromatography-mass spectrometry (LC-MS). Although these methods have high accuracy and sensitivity, they rely on expensive analytical instruments, complex pretreatment steps, and skilled professionals. In addition, these methods are not suitable for high-throughput random detection and real-time field detection. In contrast, LFIST utilizes the specific interaction between antibodies and antigens, adopts a sandwich format or a competitive format, and is both rapid and easy to use for non-professionals. LFISTs for the specific detection of bacteria, viruses, pesticides, veterinary drugs, toxins, and allergens have been developed and tested. However, there is currently no LFIST for the detection of OP. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide an immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat.

[0005] The main purpose of the present invention is to provide a preparation method of an immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat.

[0006] An immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat comprises a bottom plate, on which a sample pad, a coating reaction membrane and a water-absorbing pad which are overlapped in sequence are arranged, the coating reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area, the detection area is coated with OP-BSA coating antigen, the quality control area is coated with sheep anti-mouse IgG, and the binding layer is an anti-OP monoclonal antibody labeled with colloidal gold.

[0007] In certain embodiments, the OP-BSA is prepared by the following method:

[0008] OCA is mixed with N-hydroxysuccinimide, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride and N,N-dimethylformamide at room temperature to obtain a mixture A;

[0009] Then, the mixture A is added into PBS in which bovine serum albumin is dissolved, and stirred evenly to obtain a mixture B;

[0010] The mixture B was dialyzed against PBS and collected by centrifugation to obtain OP-BSA.

[0011] In certain embodiments, the anti-OP monoclonal antibody is obtained by the following preparation method:

[0012] 60 μg OP-BSA was mixed with 100 μL phosphate buffered saline (PBS), fully emulsified with 100 μL immune adjuvant, and then inoculated subcutaneously into the back of BALB / c female mice; Freund's complete adjuvant (FCA) was used for the first immunization, and Freund's incomplete adjuvant (FIA) was used for the second to fourth immunizations, with an interval of 21 days, for a total of 4 inoculations. After the immunization, the spleen of the immunized mouse was surgically removed, and the prepared spleen cells and myeloma cells were fused. After a series of HAT medium culture, ELISA screening, and limiting dilution subcloning, a hybridoma cell line secreting antibodies was obtained, and then the hybridoma cell line was inoculated into the peritoneal cavity of BALB / c mice to produce ascites containing antibodies, which was purified using a HiTrap Protein A column to obtain anti-OP monoclonal antibodies.

[0013] In certain embodiments, the colloidal gold-labeled anti-OP monoclonal antibody is obtained by the following preparation method:

[0014] Gold was prepared by reducing gold chloride with 1% sodium citrate (w / v), and the pH value of the colloidal gold solution was adjusted to 8.2 with 0.2 mol / L sodium carbonate; 50 μL of anti-OP mAb solution was serially diluted 2-fold in double distilled water (DDW), mixed with 200 μL of colloidal gold solution, and then 200 μL of 10% sodium chloride solution was added at room temperature to determine the optimal ratio of colloidal gold solution and anti-OP mAb solution; after 8 minutes, as the concentration of anti-OP mAb decreased, the color of the solution changed from bright red to blue; the most suitable concentration of colloidal gold-labeled mAb was the lowest concentration of the mAb solution at which the color changed; the two were mixed at the optimal ratio to obtain colloidal gold-labeled anti-OP monoclonal antibody.

[0015] In certain specific embodiments, the mass ratio of OCA, N-hydroxysuccinimide and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride is (50-60):(20-30):(40-50).

[0016] In certain specific embodiments, the mass ratio of OCA to bovine serum albumin is (50-60):(100-110).

[0017] Further: The mass ratio of OCA to bovine serum albumin is 55:105.

[0018] In certain specific embodiments, the preparation method of the coated reaction membrane comprises the following steps: using nitrocellulose membrane as the reaction membrane, adjusting the concentration of the coated antigen to 0.9-1.1 mg / mL with a coating buffer to obtain a detection coated antigen solution; and adjusting the concentration of goat anti-mouse IgG to 0.9-1.1 mg / mL with a coating buffer to obtain a quality control coated antigen solution; spraying the detection coated antigen solution and the quality control coated antigen solution onto the detection area and control area corresponding to the reaction membrane at a membrane liquid volume of 0.8-1.2 μL / cm, respectively, with the detection area and the control area being spaced 5.0 mm apart, and placing in a 45°C oven for 30 min to obtain the obtained solution; the coating buffer is a 0.1M PB buffer containing 1wt% sucrose and 0.05wt% sodium azide, with a pH of 7.4.

[0019] Compared with the prior art, the present invention has at least the following advantages:

[0020] 1) The immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat provided by the present invention comprises a bottom plate, on which a sample pad, a coating reaction membrane and a water-absorbing pad are arranged in sequence, the coating reaction membrane is a nitrocellulose membrane with a detection area and a quality control area, the detection area is coated with OP-BSA coating antigen, and the quality control area is coated with sheep anti-mouse IgG; the binding layer is an anti-OP monoclonal antibody labeled with colloidal gold. It has been confirmed that the immunochromatographic test paper in this application has a sensitivity (half inhibition concentration, IC 50 ) were 2.56 and 2.63 μg / kg, respectively, and the limit of detection (LOD) was 0.43 and 0.42 μg / kg, respectively. In terms of intra-batch and inter-batch reproducibility, the recoveries of OP spiked samples were 82.8% to 91.2%, and the coefficients of variations (CV) were less than 5.67% (intra-batch) and 6.52% (inter-batch). In parallel tests of egg samples and chicken samples, the performance of LFIST was comparable to that of high performance liquid chromatography (HPLC); the detection time of LFIST was less than 5 minutes, and it did not require reliance on professionals, so it could be used as a monitoring tool for on-site detection of OP residues, with high social and economic value.

[0021] 2) The preparation method of the present invention uses an active ester method to chemically modify oseltamivir carboxylic acid (OCA) to prepare a high-quality immunogen. After immunizing mice, the spleen cells obtained are fused with myeloma cells to obtain a monoclonal antibody with strong specificity for ELISA detection. 50 The present invention also establishes a colloidal gold immunochromatography technique for detecting oseltamivir phosphate OP in eggs / meat, which can quickly and conveniently realize the qualitative detection of oseltamivir phosphate OP in eggs / meat, and the detection sensitivity in actual samples reaches 0.42 μg / kg, with good stability, meeting the storage, transportation and use requirements in market applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0023] Figure 1 This is a schematic diagram of the assembly structure of the immunochromatographic test paper provided by the present invention;

[0024] Figure 2 for Figure 1 The immunochromatographic test shown here is a visual display of the results of the actual test procedure;

[0025] Figure 3 The immunochromatographic test paper provided by the present invention is used to detect the optical density curve of the egg sample extract;

[0026] Figure 4 The immunochromatographic test paper provided by the present invention is used to detect the optical density curve of the chicken sample extract;

[0027] Figure 5 The immunochromatographic test paper provided by the present invention is used to detect the standard curve of chicken samples;

[0028] Figure 6 The immunochromatographic test paper provided by the present invention is used to detect the standard curve of chicken samples;

[0029] Figure 7 This is a display diagram of the actual detection status of the immunochromatographic test paper provided by the present invention when used to detect chicken samples;

[0030] Figure 8 This is a display diagram of the actual detection status of the immunochromatographic test paper provided by the present invention when used to detect chicken samples. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following embodiments are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0032] When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper and lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values ​​listed herein include the endpoints of the range, and all integers and fractions within the range.

[0033] Unless otherwise indicated, all percentages, parts, ratios, etc. herein are by weight.

[0034] The materials, methods, and examples herein are illustrative and are not to be construed as limiting unless specifically stated.

[0035] In the following examples, OP and OCA were purchased from Sigma (USA); 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Fluka (China); bovine serum albumin (BSA), ovalbumin (OVA), Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were purchased from BDH (VWR International Ltd.); ethyl 4-bromobutyrate and N,N-dimethylformamide (DMF) were purchased from Aladdin Chemical Co., Ltd. (Shanghai); goat anti-mouse HRP-IgG was purchased from Sino-US Biotechnology Co., Ltd. (Luoyang, China); SPF-grade BALB / c mice were purchased from the Experimental Animal Center of Zhengzhou University and were raised in accordance with the requirements of the Animal Ethics Committee of Zhoukou Normal University; all reagents and solvents in this study were of analytical grade or higher.

[0036] Example 1

[0037] This embodiment provides a method for preparing an immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat, which specifically comprises the following steps:

[0038] 1) Preparation of immunogen OP-BSA

[0039] 58.0 mg of OCA was mixed with 26.0 mg of N-hydroxysuccinimide (NHS), 42.0 mg of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloricde [EDC] and 2 mL of N,N-dimethylformamide (DMF) at room temperature (RT) for 6 hours.

[0040] Then, the mixture was added to 3.0 mL of PBS dissolved with 107.0 mg of bovine serum albumin (BSA) and stirred at 4 °C for 12 h;

[0041] The chemical structure of the synthetic route of OP-BSA synthesized by active esterification is:

[0042]

[0043] The above solution was dialyzed against PBS, collected by centrifugation at 1510 × g for 20 min at 4°C, and then stored at −20°C to obtain OP-BSA;

[0044] 2) Preparation of coating antigen OP-OVA

[0045] 58.0 mg of OCA was mixed with 26.0 mg of N-hydroxysuccinimide (NHS), 42.0 mg of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloricde [EDC] and 2 mL of N,N-dimethylformamide (DMF) at room temperature (RT) for 6 hours.

[0046] Then, the mixture was added to 3.0 mL of PBS dissolved with 122.0 mg of chicken ovalbumin (OVA) and stirred at 4°C for 12 hours. The above solution was dialyzed against PBS, collected by centrifugation at 1510×g for 20 min at 4°C, and then stored at -20°C to obtain OP-OVA; OP-OVA was used as the coating antigen to detect the titer and sensitivity of OP polyclonal antibodies and monoclonal antibodies, respectively.

[0047] 3) Preparation of anti-OP monoclonal antibodies

[0048] 60 μg OP-BSA was mixed with 100 μL phosphate buffered saline (PBS), fully emulsified with 100 μL immune adjuvant, and then inoculated subcutaneously into the back of BALB / c female mice; Freund's complete adjuvant (FCA) was used for the first immunization, and Freund's incomplete adjuvant (FIA) was used for the second to fourth immunizations, with an interval of 21 days, for a total of 4 inoculations. After the immunization, the spleen of the immunized mouse was surgically removed, and the prepared spleen cells and myeloma cells were fused. After a series of HAT medium culture, ELISA screening, and limiting dilution subcloning, a hybridoma cell line secreting antibodies was obtained, and then the hybridoma cell line was inoculated into the peritoneal cavity of BALB / c mice to produce ascites containing antibodies, which was purified using a HiTrap Protein A column to obtain anti-OP monoclonal antibodies.

[0049] 4) Anti-OP monoclonal antibody labeled with colloidal gold

[0050] Gold was prepared by reducing gold chloride with 1% sodium citrate (w / v), and the pH value of the colloidal gold solution was adjusted to 8.2 with 0.2 mol / L sodium carbonate; 50 μL of anti-OP mAb solution was serially diluted 2-fold in double distilled water (DDW), mixed with 200 μL of colloidal gold solution, and then 200 μL of 10% sodium chloride solution was added at room temperature to determine the optimal ratio of colloidal gold solution and anti-OP mAb solution; after 8 minutes, as the concentration of anti-OP mAb decreased, the color of the solution changed from bright red to blue; the most suitable concentration of colloidal gold-labeled mAb was the lowest concentration of the mAb solution at which the color changed; the two were mixed at the optimal ratio to obtain colloidal gold-labeled anti-OP monoclonal antibody.

[0051] 5) Assembly of immunochromatographic test strips

[0052] The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially adhered to the polyethylene backing. The preparation method of the coated reaction membrane includes the following steps: using nitrocellulose membrane as the reaction membrane, adjusting the concentration of the immune antigen OP-BSA to 1.0 mg / mL with a coating buffer to obtain a detection coated antigen solution; and adjusting the concentration of goat anti-mouse IgG to 1.0 mg / mL with a coating buffer to obtain a quality control coated antigen solution; according to the membrane liquid volume of 1.0 μL / cm, the detection coated antigen solution and the quality control coated antigen solution are sprayed to the detection area and control area corresponding to the reaction membrane respectively, the interval between the detection area and the control area is 5.0 mm, and placed in a 45°C oven for 30 minutes to obtain; the coating buffer is a 0.1M PB buffer containing 1wt% sucrose and 0.05wt% sodium azide, pH=7.4.

[0053] The preparation method of the conjugate pad comprises the following steps: spraying the anti-OP monoclonal antibody labeled with colloidal gold obtained in step 4) onto the conjugate pad at 15.0 μL / cm using an XYZ3000 sprayer, and drying it in a 50°C drying oven for 30 minutes; placing the prepared conjugate pad in a plastic bag, adding a desiccant and sealing it at 4°C for storage for later use.

[0054] The assembly structure of the immunochromatographic test paper in this application is as follows Figure 1 As shown in the figure, and the visual effect diagram of the test procedure after assembly, the nitrocellulose membrane is placed on the bottom plate (polyethylene backing), the sample pad is flat on the left side of the nitrocellulose membrane, the absorbent pad is flat on the right side of the nitrocellulose membrane, and the gold label pad is flat between the sample pad and the nitrocellulose membrane, so that one end is pressed under the sample pad and the other end is covered on the nitrocellulose membrane. The overlapping length between each other is 1.5mm. After the bonding is completed, it is cut into test strips with a width of 3mm using a cutting machine; the assembled test strips are placed in a plastic card for vacuum packaging and stored at room temperature.

[0055] The working principle of the immunochromatographic test paper of the present invention is as follows: when the sample solution is loaded onto the sample pad of the LFIST, it then migrates to the adsorption layer through the capillary effect; if OP exists in the sample, it may inhibit the binding of the colloidal gold-labeled OP to the OP-BSA on the test line, thereby reducing the color intensity of the T line; within the linear range, the concentration of OP in the sample is negatively correlated with the red intensity of the T line; in any case, the control line should always appear visible red to indicate that the LFIST is effective; if the C line does not appear, the test is invalid and a new test paper should be used for retesting ( Figure 2 ).

[0056] Test Case

[0057] The present application conducts a performance test on the prepared immunochromatographic test paper, specifically:

[0058] 1. Sample preprocessing of LFIST

[0059] Chicken and egg samples from the market were pretreated and analyzed by ultra-high performance liquid chromatography (UPLC).

[0060] -MS / MS detection of the presence of OP; 3 grams of sample were homogenized in 9 mL of 0.25 mol / L ammonium acetate buffer, 9 ml of a mixture of methanol and acetonitrile (2:1, v / v) were added, shaken for 12 min, and centrifuged at 3900 × g for 5 min. 2000 μL of the supernatant was evaporated to dryness at 45 ° C under a gentle nitrogen flow. The residue was then resuspended in a mixed solution of 1000 μL PBS and methanol (4:1, V / V) and then tested.

[0061] 2. LFIST Performance Evaluation

[0062] The performance of LFIST (immunochromatographic test strip prepared in this application) was evaluated in terms of sensitivity, specificity and accuracy. The sensitivity of LFIST was determined by detecting negative chicken samples spiked with 0.0, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0 and 256.0 ng / mL OP; each sample was tested three times; in order to obtain the relative optical density (ROD) for constructing the standard curve, the color density of the T line was read with a TSR3000 membrane strip reader (Bio-Dot, USA); IC 50 is obtained from the linear regression equation.

[0063] The sensitivity was determined by testing the response of the LFIST to a series of OP-spiked samples. The relative optical density (ROD) of the T line was scanned using a Bio-Dot TSR3000 strip reader. Figure 3 , where the optical density curves from top to bottom represent 0, 1, 2, 4, 8, 16, 32, 64, 128, 256 ng / mL) and chicken samples ( Figure 4 , where the optical density curves in the figure represent 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 ng / mL from top to bottom, respectively. The ROD value is inversely proportional to the OP concentration in the sample. In both tests, the G / D×A (area) and G / peak of ROD decreased with the increase of OP concentration in the sample.

[0064] The standard OP concentration and G / D × area ROD showed an obvious linear relationship in the range of 1-32 ng / mL (for egg samples ( Figure 5 ) and chicken samples ( Figure 6 ). By dividing the G / D obtained in the standard sample by the B / B of the area-ROD 0 The percentage was plotted against the logarithmic concentration of OP in egg or chicken samples to draw a quantitative calibration curve. According to the regression equation, the IC 50 For the tested egg samples, it was 2.56 ng / mL (R 2 =0.9943), and for the tested chicken sample it was 2.63 ng / mL (R2 =0.9939).

[0065] The LOD of LFIST is quantitatively defined as the amount of OP in the standard sample solution that causes a 20% decrease in G / peak-ROD compared to the blank sample. The detection limit for egg samples was determined to be 0.43 μg / kg, and the detection limit for chicken samples was 0.42 μg / kg. In the qualitative test with the naked eye, the detection limit is determined by the minimum amount of OP that produces a significant difference between the T lines of the standard sample and the negative sample. By visual inspection, the egg sample ( Figure 7 ) and chicken samples ( Figure 8 ) reached a detection limit of 4 μg / kg.

[0066] By testing LFIST with similar competitors of OP (including amantadine, morphine, ribavirin, acyclovir, amoxicillin. 2 O and enrofloxacin) to determine its specificity. OP, OCA or competitor at a concentration of 1 μg / mL were added to negative egg samples, and then LFIST was tested. The CR calculation formula was: CR (%) = (IC 50 ) / IC of other competitors 50 )×100%.

[0067] Table 1 shows that the CR of LFIST for OP and oseltamivir carboxylic acid was 100%, and the CR for other competitors was less than 0.09%. Therefore, LFIST was highly specific for OP, and the CR for amantadine, moxifloxacin, ribavirin, acyclovir, amoxicillin trihydrate, and enrofloxacin was negligible.

[0068] Table 1. Results of cross-reactivity assay of LFIST

[0069]

[0070] To evaluate accuracy, egg and chicken samples containing 5, 10, and 20 μg / kg OP were tested with the same batch of test strips (n=6) for LFIST. For inter-batch precision, three batches of test strips were used in LFIST. Accuracy and precision were expressed as recovery and CV, respectively.

[0071] Table 2. Recovery and precision of LFIST for detection of OP added to egg and chicken samples

[0072]

[0073] Table 2 shows that for intra-batch recovery, the recovery ranged from 84.6% to 91.2%, with a maximum CV of 5.67%. For inter-batch recovery, the recovery ranged from 82.8% to 90.6%, with a maximum CV of 6.52%.

[0074] 3. Check the authenticity of LFIST

[0075] Egg and chicken samples containing 3 different concentrations of OP (3.5, 9.7, and 27.9 μg / kg, representing low, medium, and high residues) were tested by LFIST and HPLC, respectively; the results of the two methods were compared with the given concentrations by one-sample T test and analyzed by independent sample T test, respectively. The difference between the test value and the relative given concentration was considered statistically significant when P < 0.05.

[0076] Table 3 Comparison of three OP residue levels in egg and chicken samples by LFIST and HPLC

[0077]

[0078] LFIST was compared with HPLC by testing three different concentrations of OP. As shown in Table 3, there was no significant difference between the two methods in the test results of egg samples and chicken samples (P>0.05).

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. An immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat, characterized in that: The invention comprises a bottom plate, on which a sample pad, a coating reaction membrane and a water-absorbing pad which are overlapped in sequence are arranged. The coating reaction membrane is a nitrocellulose membrane provided with a detection area and a quality control area. The detection area is coated with OP-BSA coating antigen, the quality control area is coated with sheep anti-mouse IgG, and the binding layer is an anti-OP monoclonal antibody labeled with colloidal gold.

2. The immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat according to claim 1, characterized in that: The OP-BSA was prepared by the following method: OCA is mixed with N-hydroxysuccinimide, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride and N,N-dimethylformamide at room temperature to obtain a mixture A; Then, the mixture A is added into PBS in which bovine serum albumin is dissolved, and stirred evenly to obtain a mixture B; The mixture B was dialyzed against PBS and collected by centrifugation to obtain OP-BSA.

3. The immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat according to claim 2, characterized in that: The anti-OP monoclonal antibody is obtained by the following preparation method: 60 μg OP-BSA was mixed with 100 μL phosphate buffered saline solution, fully emulsified with 100 μL immune adjuvant, and then inoculated subcutaneously into the back of BALB / c female mice; Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the 2nd to 4th immunizations, with an interval of 21 days, for a total of 4 inoculations. After the immunization, the spleen of the immunized mouse was surgically removed, and the prepared spleen cells and myeloma cells were fused. After a series of HAT medium culture, ELISA screening, and limiting dilution subcloning, a hybridoma cell line secreting antibodies was obtained, and then the hybridoma cell line was inoculated into the peritoneal cavity of BALB / c mice to produce ascites containing antibodies, which was purified using a HiTrap Protein A column to obtain anti-OP monoclonal antibodies.

4. The immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat according to claim 3, characterized in that: The colloidal gold-labeled anti-OP monoclonal antibody is obtained by the following preparation method: Gold is prepared by reducing gold chloride with 1% sodium citrate (w / v), and the pH value of the colloidal gold solution is adjusted to 8.2 with 0.2 mol / L sodium carbonate; 30-80 μL of anti-OP mAb solution is diluted 2 times in double distilled water, mixed with 150-250 μL of colloidal gold solution, and then 150-250 μL of 10% sodium chloride solution is added at room temperature; as the concentration of anti-OP mAb decreases, the color of the solution changes from bright red to blue; the most suitable concentration of colloidal gold labeled anti-OP mAb is the lowest concentration of the anti-OP mAb solution at which the color changes; the two are mixed in an optimal ratio to obtain colloidal gold labeled anti-OP monoclonal antibody.

5. The immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat according to claim 2, characterized in that: The mass ratio of OCA, N-hydroxysuccinimide and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride is (50-60):(20-30):(40-50).

6. According to the preparation method of the immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat described in claim 5, the mass ratio of OCA to bovine serum albumin is (50-60): (100-110).

7. According to the method for preparing the immunochromatographic test paper for detecting oseltamivir phosphate in eggs / meat described in claim 6, the mass ratio of OCA to bovine serum albumin is 55:105.

Images