Antibody and kit for detecting citrinin in traditional Chinese medicine and application of antibody and kit

Abstract

The invention discloses an antibody and a kit for detecting citrinin in traditional Chinese medicine and application of the antibody and the kit. The rapid and accurate pretreatment method based on immunomagnetic bead design is developed, the screened citrinin antibody coupling immunomagnetic beads are applied to the method, and compared with the prior art, the operation is faster, automation is easy to achieve, the combination efficiency is high, the applicability is wide, the subsequent detection result is more accurate, and the method is suitable for large-scale popularization and application. The method is especially suitable for a traditional Chinese medicine complex matrix and an automatic high-throughput detection platform, facilitates rapid screening of citrinin in traditional Chinese medicinal materials, and can provide a simple and convenient supervision means for fungaltoxin in the traditional Chinese medicinal materials.

Description

Technical Field

[0001] This application relates to the field of analytical testing, and in particular to antibodies, kits, and their applications for detecting penicillin in traditional Chinese medicine. Background Technology

[0002] In 1995, French scholars Blanc et al. discovered that citric acid, a fungal toxin with nephrotoxicity and hepatotoxicity, is produced during the fermentation of Monascus purpureus. According to relevant literature, citric acid contamination exists in traditional Chinese medicines such as red yeast rice and areca nut, raising significant concerns about its safety. While domestic and international standards have established limits for citric acid in red yeast rice and products containing it, the 2020 edition of the Chinese Pharmacopoeia does not yet include limits for citric acid in included traditional Chinese medicines, failing to provide unified control over its content. This poses a potential safety risk from fungal toxins. Therefore, it is necessary to establish efficient and accurate detection methods to quantitatively analyze citric acid in traditional Chinese medicines to ensure clinical medication safety.

[0003] Traditional Chinese medicine (TCM) materials have complex matrices and contain numerous interfering components, thus limiting the methods for detecting citric acid penicillin. Currently, commonly used methods for citric acid penicillin detection mainly include colorimetry, thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), and high-performance liquid chromatography (HPLC). While colorimetry is rapid and inexpensive, its sensitivity is low and recovery rates are unsatisfactory under certain experimental conditions. TLC is simple, requires minimal equipment, and is easy to implement, but it cannot achieve accurate quantification and is susceptible to interference from various factors. ELISA involves simple sample pretreatment, low solvent consumption, and a short detection cycle, but it carries the risk of false positives, affecting the reliability of the results.

[0004] Because citric acid exhibits natural fluorescence, high-performance liquid chromatography-fluorescence detection (HPLC-FLD) can achieve sensitivity close to that of liquid chromatography-mass spectrometry (LC-MS) while requiring less technical expertise from operators. Current HPLC-FLD-based methods for citric acid detection include direct extraction, solid-phase extraction, and immunoaffinity column methods. However, these methods still have limitations in accuracy and sensitivity; some methods have recoveries below 70% and relatively high limits of detection. Existing sample pretreatment methods are ineffective in reducing matrix effects, time-consuming, and cumbersome. Summary of the Invention

[0005] The purpose of this application is to provide an antibody against penicillin.

[0006] Another objective of this application is to provide a kit for detecting penicillin in traditional Chinese medicine.

[0007] Another objective of this application is to provide a method for detecting penicillin in traditional Chinese medicine.

[0008] To address the aforementioned technical problems, a first aspect of this application provides an antibody, the antibody comprising: Light chain and heavy chain; wherein the light chain includes a light chain variable region VL, the light chain variable region including CDRL1 as shown in SEQ ID No:1, CDRL2 as shown in SEQ ID No:1 and CDRL3 as shown in SEQ ID No:1; The heavy chain includes a heavy chain variable region VH, which includes CDRH1 as described in SEQ ID No:6, CDRH2 as described in SEQ ID No:7, and CDRH3 as described in SEQ ID No:8.

[0009] In some preferred embodiments, the light chain variable region VL is as shown in SEQ ID No:4.

[0010] In some preferred embodiments, the light chain is as shown in SEQ ID No:5.

[0011] In some preferred embodiments, the heavy chain variable region VH is as shown in SEQ ID No:9.

[0012] In some preferred embodiments, the heavy chain is as shown in SEQ ID No:10.

[0013] In some preferred embodiments, the antibody is a murine antibody or a human antibody.

[0014] In some preferred embodiments, the antibody is a monoclonal antibody.

[0015] In some preferred embodiments, the antibody is an anti-citric acid antibody.

[0016] In some preferred embodiments, the antibody is used to detect penicillin in traditional Chinese medicine.

[0017] In some preferred embodiments, the IC50 of the hybridoma cell line 50 The value should not exceed 2.0 ng / mL.

[0018] In a second aspect, the present invention provides a kit containing immunomagnetic beads, the immunomagnetic beads being coupled with the antibody described in the first aspect of the present invention.

[0019] In some preferred embodiments, the kit is an immunomagnetic bead kit.

[0020] In some preferred embodiments, the kit includes: (i) Immunomagnetic beads, wherein the immunomagnetic beads are coupled with the antibody described in the first aspect of the present invention; (ii) Buffer system; and (iii) Quality control products.

[0021] In some preferred embodiments, the immunomagnetic beads are magnetic iron oxide microspheres coupled with the antibody described in the first aspect of the present invention.

[0022] In some preferred embodiments, the magnetic iron oxide microspheres have a particle size of 20–60 μm.

[0023] In some preferred embodiments, the surface of the magnetic iron oxide microspheres is coated with an agarose polymer layer.

[0024] In some preferred embodiments, the immunomagnetic beads are prepared by the following method: taking immunomagnetic bead microspheres activated with hydrogen bromide, adding a coupling buffer (e.g., phosphate buffer at pH 7) to reconstitute and obtain swollen immunomagnetic bead microspheres; then taking the swollen immunomagnetic bead microspheres and mixing them with the antibody described in the first aspect of the present invention and stirring to react, so that the antibody is coupled to the microspheres, and then using a blocking agent to block them, thereby obtaining immunomagnetic bead microspheres coupled with the antibody described in the first aspect of the present invention.

[0025] In some preferred embodiments, the buffer system includes a binding buffer, a washing buffer, and an elution buffer.

[0026] In some preferred embodiments, the binding solution is a PBS eluent containing 0.1% polysorbate.

[0027] In some preferred embodiments, the washing solution is a 5% aqueous solution of methanol.

[0028] In some preferred embodiments, the eluent is a mixture of acetonitrile and 0.1% phosphoric acid solution; for example, 0.5 ml acetonitrile: 0.1% phosphoric acid solution (7:3).

[0029] In some preferred embodiments, the kit includes separate first, second, third, and fourth wells, wherein the first well contains a binding solution, the second well contains a binding solution, the third well contains a washing solution, and the fourth well contains an elution solution. Preferably, the test sample is passed through the first, second, third, and fourth wells sequentially during the reaction.

[0030] In some preferred embodiments, the quality control materials include negative controls and positive controls.

[0031] A third aspect of the present invention provides a method for detecting penicillin in traditional Chinese medicine, comprising the steps of: using the reagent kit described in the second aspect of the present invention to detect the sample to be tested.

[0032] In some preferred embodiments, the method includes the steps of: (1) Pass the sample to be tested through the first well, the second well, the third well, the first well and the fourth well in sequence. The first well contains binding solution, the second well contains binding solution, the third well contains washing solution and the fourth well contains elution solution; collect the elution solution in the fourth well, and then make up to volume with ammonia solution to obtain the test solution; (2) Use liquid chromatography to detect the test solution.

[0033] In some preferred embodiments, the chromatographic conditions for the liquid chromatography include: Use 0.1% phosphoric acid:acetonitrile (V / V=55:45) as the mobile phase; Phenomenex Luna C18(2) chromatographic column; Excitation wavelength λ of fluorescence detector ex =331nm, emission wavelength λ em =500nm; The column temperature is 30°C; and / or The flow rate was 1 ml per minute.

[0034] In some preferred embodiments, the traditional Chinese medicine is selected from red yeast rice or areca nut.

[0035] Compared with the prior art, the present invention has at least the following advantages: The present invention develops a rapid and accurate pretreatment method based on immunomagnetic beads. This method uses antibodies of screened penicillin conjugated with immunomagnetic beads. Compared with the prior art, the operation is faster, easier to automate, has high binding efficiency, and wide applicability, making the subsequent detection results more accurate. It is especially suitable for complex matrices of traditional Chinese medicine and automated high-throughput detection platforms, which can help to rapidly screen penicillin in traditional Chinese medicine and provide a simple means of monitoring mycotoxins in traditional Chinese medicine.

[0036] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0037] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0038] Figure 1 This is a chromatogram of a red yeast rice sample according to an embodiment of the present invention; Figure 2 This is a chromatogram of a areca nut sample according to an embodiment of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0040] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0041] Example 1: Preparation and screening of penicillin antibodies (1) Animal immunization In this study, BALB / c mice were immunized five times with two-week intervals between each immunization using CIT-BSA and CIT-KLH immunogens. The inhibitory levels of citric acid in the serum of mice after the five immunizations were measured using an indirect competitive ELISA. The results showed that both CIT-BSA and CIT-KLH immunogens had good immunizing effects, with CIT-KLH exhibiting better inhibitory effects and a higher titer. Therefore, mice 4 (IC50-7000) immunized with CIT-KLH were selected for this experiment.

[0042] (2) Cell fusion Take samples containing 6×10 7 Spleen cells from CIT-KLH-immunized mouse No. 4 and containing 6×10 6Cell suspensions of SP2 / 0 cells were combined and placed in a 50 mL plastic centrifuge tube. Centrifuge at 1000 rpm for 5 min, discarding the supernatant. Gently tap the bottom of the tube with your finger to loosen the precipitate into a paste. Take 1 mL of 50% PEG 1500. While rotating the centrifuge tube at a constant speed with your left hand, use a 1 mL pipette in your right hand to slowly add 0.7 mL of the 50% PEG solution along the rotating tube wall (as close to the cells as possible), completing the addition within 60 seconds. Then, slowly aspirate the cell suspension into the pipette, completing aspiration within 30 seconds. After standing for 30 seconds, gently blow the cell suspension back into the centrifuge tube within 30 seconds. Immediately add 25 mL of DMEM incomplete culture medium within 5 min to terminate the reaction. Centrifuge at 1000 rpm for 7 min, discarding the supernatant. Add 20 mL of HAT culture medium and gently aspirate to suspend the precipitated cells. Add the cell suspension to a 96-well cell culture plate already seeded with feeder cells. mL / well, incubated in an incubator at 37℃ with 5% CO2; on the 3rd day after fusion, half of the medium in each well was replaced with HAT complete culture medium, and on the 7th and 10th days after fusion, half of the medium was replaced with HT complete culture medium, and thereafter the medium was replaced with complete culture medium.

[0043] (3) Screening and cloning of hybridoma cells When the fused cell clones grew to 1 / 4-1 / 3 of the well bottom area, the culture supernatant from all clone growth wells was detected using an indirect competitive ELISA method. Cells from wells with high OD values, high inhibition rates, and fewer clones were selected for cloning and expanded culture. After 3-4 subclonings, hybridoma cell lines with the highest affinity and stable secretion of anti-penicillin monoclonal antibodies were obtained. In this experiment, five cell lines were cloned: 1, 2, 3, 4, and 5. The IC50 values ​​for these five cell lines were... 50 The values ​​were 2.2 ng / mL, 1.2 ng / mL, 2.9 ng / mL, 4.8 ng / mL, and 3.6 ng / mL, respectively, with cell line 2 showing the best inhibition. Cell line 2 is the cell line containing the anti-penicillin monoclonal antibody.

[0044] (4) Preparation of monoclonal antibodies Hybridoma cells (cell 2) selected from the screening were cultured in large quantities and injected into the peritoneal cavity of female BALB / c mice. Slight abdominal distension was observed on day 6, and significant abdominal enlargement was observed on days 8-10. Ascites fluid was aspirated using a syringe, with 4-10 mL collected from each mouse per aspiration. After centrifugation, the supernatant was pale yellow. The supernatant was collected and purified using the saturated ammonium sulfate method to obtain antibodies. The purified monoclonal antibody protein was diluted and its OD value was measured using a UV spectrophotometer. The formula was: Protein concentration (mg / mL) = 1.45 × OD. 280nm -0.74×OD 260nm The antibody concentration prepared in this experiment was 8.4 mg / ml. (5) Purification of monoclonal antibodies Monoclonal antibodies from ascites fluid were purified using the octanoic acid-saturated ammonium sulfate method. Five mL of ascites fluid was taken, and 10 mL of 0.06 mol / L, pH 5.0 acetate buffer was added. The pH was adjusted to 4.8 with 0.1 mol / L HCl. 160 μL of octanoic acid was added dropwise under stirring at room temperature. The mixture was allowed to stand at 4 °C for 2 h, then centrifuged at 8000 rpm for 10 min, and the precipitate was discarded. Two mL of 0.1 mol / L PBS was added to the supernatant, and the pH was adjusted to 7.4 with 1 mol / L NaOH. A suitable amount of saturated ammonium sulfate was added dropwise to achieve 45% saturation, and the mixture was allowed to stand for 1 h. The mixture was centrifuged at 8000 rpm for 30 min at 4 °C, and the supernatant was discarded. The precipitate was dissolved in 5 mL of pH 7.4 PBS, dialyzed for 2 days, centrifuged again, aliquoted, and stored at -20 °C for later use.

[0045] (6) Antibody sequencing After pretreatment, the monoclonal antibody samples were hydrolyzed into short peptides using multiple enzymes. These peptides were then detected using a high-precision mass spectrometer. A de novo sequencing algorithm was then used to convert the tandem mass spectrometry data into short peptide sequences, which were finally assembled back into complete antibody protein sequences. The sequencing results of the penicillin antibody prepared in this embodiment are as follows: (CDRL1) RANKNLDRYGI (SEQ ID No:1) (CDRL2) LLIFAAS (SEQ ID No:2) (CDRL3) CQQSKEVPPT(SEQ ID No:3) (VL) DIVLTQSPASLAVSLGQRATISCRANKNLDRYGISFVNWFQQKQGQPPKLLIFAASNQGSGVPAKFSGSGSGTDFSLNIHPVDEDDTALYFCQQSKEVPPTFGGGTRLEL(SEQ ID No:4) (Light sequence) DIVLTQSPASLAVSLGQRATISCRANKNLDRYGISFVNWFQQKQGQPPKLLIFAASNQGSGVPAKFSGSGSGTDFSLNIHPVDEDDTALYFCQQSKEVPPTFGGGTRLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID No:5) (CDRH1) SSFGM(SEQ ID No:6) (CDRH2) YISGDSSTIYYKDTVK(SEQ ID No:7) (CDRH3) TRAPLFGNYVVDY(SEQ ID No:8) (VH) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISGDSSTIYYKDTVKGRVTISRDNPRNTLFLQMTSLRSEDTAIYYCTRAPLFGNYVVDYWGQGTTLTVSS(SEQ ID No:9) (Heavy sequence) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISGDSSTIYYKDTVKGRVTISRDNPRNTLFLQMTSLRSEDTAIYYCTRAPLFGNYVVDYWGQG TTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTIN PCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPI ERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPG (SEQ ID No:10) (7) Antibody specificity Indirect competitive ELISA was used to serially dilute fungal toxin drugs, and concentration-inhibition rate curves were plotted based on the detection results to calculate the IC50 of each competitor. 50 The values ​​were used to examine the specific reactions of the antibodies against other fungal toxins. The results (Table 2) showed that the screened penicillin antibodies did not show specific reactions against any other fungal toxins.

[0046] Table 2. Antibody-specific response status

[0047] Example 2: Preparation of the detection system This application uses a penicillin immunomagnetic bead kit, which includes: (1) The immunomagnetic beads are magnetic iron oxide microspheres activated with cyanogen bromide. The particle size of the magnetic microspheres is 20-60 μm. The magnetic microspheres are rich in an agarose polymer layer (purchased from Beijing Shuntongyun Technology Co., Ltd.). The preparation steps include coupling and blocking. Conjugation: 0.1 mol / L hydrochloric acid solution was added to 1.0 g of hydrogen bromide-activated immunomagnetic beads, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded; phosphate buffer at pH 7 was added for reconstitution to obtain swollen magnetic bead microspheres; then the prepared penicillin monoclonal antibody was added to the swollen magnetic bead microspheres, and the mixture was thoroughly mixed at 25 °C using an end-over-end method for 12 h, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded to obtain the magnetic bead microsphere matrix.

[0048] Blocking: Transfer the magnetic bead microsphere matrix to 0.1 mol / L blocking reagent, let it stand at 25℃ for 2-4 h, centrifuge at 2000 rpm for 10 min, discard the supernatant, and finally reconstitute with PBS washing buffer to obtain the product.

[0049] (2) The coupling buffer solution is a phosphate buffer with a pH of 7. The specific preparation method is as follows: weigh 8.0g sodium chloride, 1.2g disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, and 0.2g potassium chloride, add 990ml of water to dissolve, adjust the pH value to 7.0 with hydrochloric acid, and dilute with water to 1000ml.

[0050] (3) The blocking reagent is PBS rinsing solution with pH 7.0. The specific preparation method is to weigh 8.0g sodium chloride, 1.2g disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, and 0.2g potassium chloride, add 990ml of water to dissolve, adjust the pH value to 7.0 with hydrochloric acid, and dilute with water to 1000ml.

[0051] (4) The kit consists of four wells: a binding buffer well, a reaction well 1, a reaction well 2, and an elution buffer well. The binding buffer well contains 10 ml of 0.1% polysorbate-20 PBS elution buffer, reaction well 1 contains 1 ml of 0.1% polysorbate-20 PBS elution buffer, reaction well 2 contains 1 ml of 5% methanol aqueous solution, and the elution buffer well contains 0.5 ml of acetonitrile:0.1% phosphate solution (7:3). The PBS elution buffer is prepared by weighing 8.0 g sodium chloride, 1.2 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, and 0.2 g potassium chloride, dissolving them in 990 ml of water, adjusting the pH to 7.0 with hydrochloric acid, and diluting with water to 1000 ml.

[0052] Example 3, Detection Method (1) Preparation of standard curve Accurately weigh an appropriate amount of penicillin reference standard and add 70% methanol to prepare a solution containing 100 ng per ml, which will be used as a stock solution. Take an appropriate amount of the stock solution and prepare solutions containing 0.5 ng, 1 ng, 2 ng, 5 ng, 10 ng, and 20 ng per ml with 70% methanol to obtain a series of reference standard solutions.

[0053] (2) Sample preparation Accurately weigh 1g of sample powder, add 20ml of 70% methanol, weigh again, vortex for 1 minute, and shake in an 80℃ water bath (150rpm) for 30 minutes. Cool, replenish the lost weight with 70% methanol, mix well, centrifuge (3900 rpm) for 5 minutes, accurately pipette 0.5ml of the supernatant into the binding well of the immunomagnetic bead kit, mix well, add 800μl of penicillin immunomagnetic beads, react for 20 minutes, and after magnetic separation by magnetic rod for 30 seconds, transfer the immunomagnetic beads to well 1 of the reaction tube for rinsing. After 1 minute of separation by magnetic attraction with a magnetic rod for 30 seconds, the immunomagnetic beads are transferred to reaction well 2 and rinsed for 1 minute. After 30 seconds of separation by magnetic attraction with a magnetic rod, the immunomagnetic beads are transferred to the binding well and eluted for 5 minutes. After 40 seconds of separation by magnetic attraction with a magnetic rod, the immunomagnetic beads are discarded, and the eluent in the elution well is collected. The eluent is diluted with ammonia solution (1 ml concentrated ammonia solution → 1 L water) to a volumetric flask and shaken well. The flask is then centrifuged at high speed (9500 rpm) for 5 minutes, and the supernatant is collected.

[0054] (3) Liquid chromatography method The chromatographic column was an octadecylsilane-bonded silica gel, with a Phenomenex Luna C18(2) column (25 cm long, 4.6 mm inner diameter, and 5 μm particle size); the mobile phase was 0.1% phosphoric acid:acetonitrile (55:45); and the detection was performed using a fluorescence detector with an excitation wavelength of λ. ex =331nm, emission wavelength λ em The column temperature was 30℃ at 500 nm; the flow rate was 1 ml per minute. 50 μl of each of the series of reference solutions and sample solutions were injected into the high-performance liquid chromatograph for analysis.

[0055] 3.1 Linearity: 50 μl of each of the citric acid reference solutions with concentrations of 0.25, 0.5, 1, 2, 5, 10, and 20 ng / ml were precisely pipetted into the liquid chromatograph for analysis. Peak areas were measured, and a standard curve was plotted with peak area as the ordinate and concentration (ng / ml) as the abscissa. The regression equation was: y = 16974.9*x - 1598.98, with a correlation coefficient of 0.9997924, indicating that the method exhibits good linearity in the range of 0.25 ng / ml to 20 ng / ml.

[0056] 3.2 Accuracy: 1g of negative samples of red yeast rice and areca nut were accurately weighed, in nine portions (three portions each of low, medium, and high concentrations). 500μl, 1000μl, and 2500μl of penicillin reference standard stock solution (concentration 100 ng / ml) were accurately added to each portion (spiking levels of 50 μg / kg, 100 μg / kg, and 250 μg / kg, respectively). The sample solution was prepared according to the prescribed method. The recoveries at the high, medium, and low concentrations were investigated. The results (Table 2-3) showed that the recoveries of both red yeast rice and areca nut matrices were 70-120%, and the RSDs were all less than 10%, indicating good accuracy of the method.

[0057] Table 2. Recovery rate results of red yeast rice samples

[0058] Table 3. Recovery rate results of areca nut samples

[0059] 3.3 Repeatability: Six copies of each of the red yeast rice and areca nut positive samples were accurately weighed and prepared according to the procedure under the test solution preparation section. The average value and RSD of the six copies were calculated. The results (Table 4-5) showed that the RSD of penicillin detection in both red yeast rice and areca nut matrices was less than 10%, indicating good repeatability of the method.

[0060] Table 4. Repeatability Results of Red Yeast Rice

[0061] Table 5. Repeatability results of areca nut

[0062] 3.4 Limit of Detection and Limit of Quantification: Taking the sample at the low concentration loading point (i.e., the loading level is 50 μg / kg) under the accuracy test, the test solution was prepared according to the proposed test solution preparation method. The signal-to-noise ratio was measured, and the method limit of detection and limit of quantification were calculated using signal-to-noise ratios of 3:1 and 10:1. The method limit of detection was 1.3 μg / kg, and the limit of quantification was 4.3 μg / kg, indicating that the method has high sensitivity.

[0063] Table 5 Results of Limit of Detection and Limit of Quantification

[0064] Example 4 Red yeast rice powder was taken and pretreated according to the method provided in this invention. Then, it was analyzed by high-performance liquid chromatography (HPLC) to obtain the content of penicillin in the red yeast rice sample. The detection results of three batches of samples are shown in Table 6. Figure 1 .

[0065] Table 6 Detection results of red yeast rice samples

[0066] Example 5 Areca nut powder was taken, and the sample was pretreated according to the method provided in this invention. Then, it was analyzed by high performance liquid chromatography to obtain the content of penicillin in the areca nut sample. The detection results of three batches of samples are shown in Table 7. Figure 2 .

[0067] Table 7 Detection results of areca nut samples

[0068] Those skilled in the art will understand that the above embodiments are specific implementations of this application, and in practical applications, various changes can be made in form and detail without departing from the spirit and scope of this application.

Claims

1. An antibody, characterized in that, The antibodies include: Light chain and heavy chain; wherein the light chain includes a light chain variable region VL, the light chain variable region including CDRL1 as shown in SEQ ID No:1, CDRL2 as shown in SEQ ID No:1 and CDRL3 as shown in SEQ ID No:1; The heavy chain includes a heavy chain variable region VH, which includes CDRH1 as described in SEQ ID No:6, CDRH2 as described in SEQ ID No:7, and CDRH3 as described in SEQ ID No:

8.

2. The antibody according to claim 1, characterized in that, The light chain variable region VL is as shown in SEQ ID No:4; and / or the heavy chain variable region VH is as shown in SEQ ID No:

9.

3. The antibody according to claim 1, characterized in that, The light chain is shown in SEQ ID No:5; And / or; the heavy chain is as shown in SEQ ID No:

10.

4. The antibody according to claim 1, characterized in that, The antibody is a monoclonal antibody.

5. An immunomagnetic bead reagent kit, characterized in that, The kit includes immunomagnetic beads, wherein the immunomagnetic beads are conjugated with an antibody as described in any one of claims 1-4.

6. The immunomagnetic bead reagent kit according to claim 5, characterized in that, The immunomagnetic bead kit includes: (i) Immunomagnetic beads, wherein the immunomagnetic beads are coupled with an antibody as described in any one of claims 1-4; (ii) Buffer system; and (iii) Quality control products.

7. The immunomagnetic bead reagent kit according to claim 6, characterized in that, The immunomagnetic beads are magnetic iron oxide microspheres; Preferably, the magnetic iron oxide microspheres have a particle size of 20–60 μm; Preferably, the surface of the magnetic iron oxide microspheres is coated with an agarose polymer layer.

8. The immunomagnetic bead reagent kit according to claim 6, characterized in that, The buffer system includes a binding buffer, a washing buffer, and an elution buffer; wherein... The binding solution is PBS rinsing solution, which contains 0.1% polysorbate; And / or, the eluent is a mixture of acetonitrile and 0.1% phosphoric acid solution; And / or, the washing solution is a 5% aqueous solution of methanol.

9. A method for detecting penicillin in traditional Chinese medicine, characterized in that, The method includes the step of: detecting the sample to be tested using the kit as described in any one of claims 5-8; preferably, the method includes the step of: (1) Pass the sample to be tested through the first well, the second well, the third well, the first well and the fourth well in sequence. The first well contains binding solution, the second well contains binding solution, the third well contains washing solution and the fourth well contains elution solution; collect the elution solution in the fourth well, and then make up to volume with ammonia solution to obtain the test solution; (2) Use liquid chromatography to detect the test solution.

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