Colloidal gold immunochromatographic test strip for detecting melilotus suaveolens and its preparation method and application

Abstract

The present application relates to the fields of biotechnology and food detection technology, and discloses a rice flower honey colloidal gold immunochromatographic test strip as well as a preparation method and application thereof. Through proteomic analysis, the Ycf1 protein of chloroplast origin is identified as a specific marker of the plant source in rice flower honey, and a rice flower honey colloidal gold immunochromatographic test strip is developed. The test strip comprises a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are connected in sequence. The gold label pad is coated with gold-labeled antibodies formed by the combination of a monoclonal antibody against a specific segment of the Ycf1 protein and colloidal gold. The nitrocellulose membrane is provided with a detection line and a quality control line. The detection line is coated with a monoclonal antibody against the Ycf1 protein, and the quality control line is coated with a goat anti-mouse IgG. The test strip is simple to operate, and the detection can be completed within 15 minutes. The test strip has high sensitivity, strong specificity, and the results are intuitive and reliable, and is suitable for the on-site rapid screening and identification of the authenticity of rice flower honey.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and food testing technology, and more specifically, to a colloidal gold immunochromatographic test strip for rice honey, its preparation method, and its application. Background Technology

[0002] Leucosceptrum honey is a natural sweet substance produced by Chinese honeybees from the nectar of the Leucosceptrum flower, which is then mixed with their own secretions and fully fermented. Leucosceptrum honey has a very distinctive black color, hence its alternative name, "black honey." As an important specialty honey from Chinese honeybees, its nectar source, Leucosceptrum, belongs to the genus Leucosceptrum in the family Lamiaceae. It grows as a shrub or small tree, flowering from February to April each year. Due to climatic conditions, Leucosceptrum honey has a long production cycle and high maturity, making it a high-end honey product. Currently, common methods for identifying the plant source of honey include sensory evaluation, palynological analysis, and mass spectrometry. While the results are accurate and reliable, the testing equipment is expensive, the analytical procedures are complex, require professional personnel, and sample pretreatment is cumbersome. The high cost and long cycle of a single test make it unsuitable for rapid screening and large-scale testing. Therefore, there is an urgent need to develop a method for identifying Leucosceptrum honey that combines laboratory-level accuracy with the convenience of rapid on-site testing. Summary of the Invention

[0003] The purpose of this invention is to provide a colloidal gold immunochromatographic test strip for rice flower honey, its preparation method, and its application.

[0004] To achieve the objectives of this invention, in a first aspect, the invention provides any of the following applications of the Ycf1 protein or a truncated form thereof:

[0005] 1) Used for the identification of rice flower honey;

[0006] 2) As a characteristic marker of rice flower honey;

[0007] 3) Used to construct the fingerprint spectrum of rice flower honey;

[0008] The Ycf1 protein is:

[0009] (A) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0010] (B) A protein derived from (A) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:2 and having the same function;

[0011] Preferably, the truncated version of the Ycf1 protein is the amino acid sequence from position 255 to 698 of the Ycf1 protein;

[0012] More preferably, the truncated form of the Ycf1 protein is:

[0013] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or

[0014] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0015] Secondly, the present invention provides a method for identifying the authenticity of rice flower honey. The method involves detecting the components in the honey to be tested. If the honey contains Ycf1 protein or its truncated form, the honey to be tested is determined to be rice flower honey or adulterated with rice flower honey.

[0016] Thirdly, the present invention provides a monoclonal antibody against Ycf1 protein, wherein the monoclonal antibody is secreted by hybridoma cell line A11 with accession number CGMCC No. 46751 or hybridoma cell line C9 with accession number CGMCC No. 46752.

[0017] Fourthly, the present invention provides a composition comprising a monoclonal antibody secreted by hybridoma cell line A11 with accession number CGMCC No. 46751 and a monoclonal antibody secreted by hybridoma cell line C9 with accession number CGMCC No. 46752.

[0018] Fifthly, the present invention provides any of the following applications of the monoclonal antibody or the composition:

[0019] (i) Used to detect Ycf1 protein or its truncated form in samples;

[0020] (ii) Prepare reagents, test strips or kits for detecting Ycf1 protein or its truncated form.

[0021] In a sixth aspect, the present invention provides a colloidal gold immunochromatographic test strip for rice flower honey, comprising a sample pad, a gold label pad, a nitrocellulose membrane and an absorbent pad stacked in sequence;

[0022] The gold-labeled pad is coated with a gold-labeled antibody formed by the combination of a first antibody and colloidal gold; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with a second antibody, and the control line is coated with goat anti-mouse IgG.

[0023] Wherein, the first antibody is a monoclonal antibody secreted by hybridoma cell line A11 with accession number CGMCC No. 46751, and the second antibody is a monoclonal antibody secreted by hybridoma cell line C9 with accession number CGMCC No. 46752; or,

[0024] The first antibody is a monoclonal antibody secreted by hybridoma cell line C9 with accession number CGMCC No.46752, and the second antibody is a monoclonal antibody secreted by hybridoma cell line A11 with accession number CGMCC No.46751.

[0025] Hybridoma cell lines A11 and C9 are now deposited at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession numbers CGMCC No. 46751 and CGMCC No. 46752, respectively, and the deposit date is November 14, 2025.

[0026] Preferably, the sample pad is a glass fiber membrane.

[0027] In a seventh aspect, the present invention provides a method for preparing the test strip, comprising the following steps:

[0028] (1) Prepare colloidal gold solution;

[0029] (2) Add the purified first antibody to the colloidal gold solution and stir to make the first antibody stably bind to the colloidal gold particles;

[0030] (3) Add bovine serum albumin (BSA) solution for blocking, purify by high-speed centrifugation, resuspend in buffer containing stabilizer to prepare gold pad working solution;

[0031] (4) Spray the gold-labeled pad working solution evenly onto the glass fiber membrane, dry it, and prepare the gold-labeled pad;

[0032] (5) The second antibody is streaked onto a nitrocellulose membrane at a certain concentration to form a detection line;

[0033] (6) Sheep anti-mouse IgG antibody was streaked onto a nitrocellulose membrane at a certain concentration to form a quality control line;

[0034] (7) Stack the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC backing in sequence, and cut them into test strips of appropriate width.

[0035] Eighthly, the present invention provides the application of the test strip in the detection of rice flower honey.

[0036] Ninthly, the present invention provides a method for detecting rice flower honey using the test strip, the method comprising: diluting the honey sample to be tested with water, inserting the test strip, allowing the diluted honey sample to saturate the sample pad, and reading the result after a period of time;

[0037] The results are judged as follows: when the control line is red, the result is valid; when the test line shows color, the honey sample is determined to be honey from rice flower or honey from rice flower that has been mixed in; when the control line does not show color, the result is invalid.

[0038] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0039] (I) The colloidal gold immunochromatographic test strip provided by this invention is suitable for rapid and accurate detection of the authenticity of honey in rice flower. Colloidal gold immunochromatographic test strip technology is a novel colloidal gold-labeled immunochromatographic technology with the characteristics of visualization, high specificity, speed and convenience. It can be used for indirect qualitative or semi-quantitative detection, with low requirements for testing personnel, and is very suitable for rapid on-site detection of honey.

[0040] (ii) The present invention prepares a monoclonal antibody specific to the Ycf1 segment of *Milanhua* through monoclonal antibody preparation technology. It has the advantages of high sensitivity and strong specificity, and has no cross-reaction with other related proteins.

[0041] (III) The colloidal gold test strip for identifying honey species in rice flower of the present invention has the advantages of convenient operation and short detection time, and is suitable for identifying the authenticity of bee species in rice flower honey. Attached Figure Description

[0042] Figure 1 This is an SDS-PAGE electrophoresis image of bacterial cells expressed in large quantities in a preferred embodiment of the present invention.

[0043] Figure 2 This is an example of a colloidal gold test strip for honey in rice flower prepared according to a preferred embodiment of the present invention, along with the criteria for determining the test results.

[0044] Figure 3 This is a graph showing the specificity determination results of the colloidal gold test strip for honey in rice flower as described in Example 4 of the present invention.

[0045] Figure 4 This is a graph showing the repeatability test results of the colloidal gold test strip for honey in rice flower as described in Example 5 of the present invention.

[0046] Figure 5 This is a graph showing the sensitivity determination results of the colloidal gold test strip for honey in rice flower as described in Example 6 of the present invention. Detailed Implementation

[0047] One objective of this invention is to provide a characteristic marker protein Ycf1 of rice flower honey and its 255-698 amino acid sequence as a specific antigenic region.

[0048] The second objective of this invention is to provide an antigen preparation and screening scheme using a specific region of the Ycf1 protein.

[0049] The third objective of this invention is to provide a colloidal gold test strip for rapid identification of rice flower honey, which is prepared based on the aforementioned Ycf1 protein specific region.

[0050] The present invention adopts the following technical solution:

[0051] This invention provides a rapid identification method for honey from *Rhizophora stylosa*, particularly concerning the development and application of the specific protein Ycf1 encoded by the chloroplast genome of *Rhizophora stylosa* and its derived peptides as characteristic markers for identifying honey from *Rhizophora stylosa*. This invention is based on the protein identification results of honey from *Rhizophora stylosa* using liquid chromatography-tandem mass spectrometry, discovering a characteristic marker for identifying honey from *Rhizophora stylosa* (…). Leucosceptrum canum The chloroplast genome-encoded specific protein Ycf1 has relatively high abundance and is stably present in all honey samples from *Rhizophora stylosa*. Its full sequence underwent transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disordered sequence analysis, antigenicity analysis, homology analysis, and domain analysis. Ultimately, the amino acid sequence described in SEQ ID NO:1 was selected as the antigen sequence for expression and preparation of the antigen in *E. coli*.

[0052] This invention further purifies the prepared Ycf1 recombinant protein and immunizes Balb / c mice, obtaining positive cell lines through cell fusion technology. After multiple rounds of subclonal screening and specificity verification, and excluding cross-reactivity with other plant proteins, nine Ycf1-specific monoclonal cell lines were obtained. The five cell lines with the highest affinity were selected for antibody epitope pairing experiments. The final determined antibody pairs exhibited excellent linearity and high sensitivity, and were used to construct a colloidal gold immunochromatographic test strip for detecting *Milania micrantha* honey.

[0053] The colloidal gold immunochromatographic test strip of this invention comprises, in sequence, a gold-labeled pad, an NC membrane containing a test line and a control line, a sample pad, an absorbent pad, and a test base plate. The gold-labeled pad contains a conjugate of a specific segment monoclonal antibody against *Ycf1* sp. *rice* and colloidal gold; the NC membrane contains a test line (T line) and a control line (C line); the test line is composed of a specific segment monoclonal antibody against *Ycf1* sp. *rice* sp. *rice*, and the control line is composed of goat anti-mouse IgG antibody.

[0054] The present invention also provides the application of the colloidal gold test strip in the authenticity detection of honey from rice flower, the application including the detection of the authenticity of the source of honey from rice flower and the detection of adulteration of honey from rice flower with other types of honey.

[0055] The application of this invention is achieved through the following steps:

[0056] ① Remove the test strip and allow it to return to room temperature;

[0057] ② Preparation of honey sample detection solution: Dilute the honey sample with pure water at a mass ratio of 2:5 and mix well;

[0058] ③ Insert the test strip into the sample tube to allow the diluted honey sample to fully soak the sample pad, and read the result after 15 minutes;

[0059] ④ Result judgment: When the C line shows a red line, the result is valid. When the T line shows color, the result is that the honey in Leucosceptrum canum or the honey mixed with Leucosceptrum canum, that is, the tested sample contains Leucosceptrum canum Ycf1 protein. When the C line does not show color, the result is invalid.

[0060] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0061] Example 1 Isolation and purification of Leucosceptrum canum Ycf1 protein antigen

[0062] I. Selection of recombinant protein

[0063] The proteins with relatively high abundances identified in the honey of Leucosceptrum canum are shown in Table 1.

[0064] Table 1 List of main proteins identified in the honey of Leucosceptrum canum

[0065]

[0066] Based on the identification results of proteins in honey, the specific protein Ycf1 encoded by the Leucosceptrum canum chloroplast genome has relatively high contents and is stable in the honey of Leucosceptrum canum, and exists only in the honey of Leucosceptrum canum. Therefore, the Ycf1 protein was selected as the reference protein for site mutation and recombined using Escherichia coli. The amino acid sequence of the Ycf1 protein is shown in SEQ ID NO:2.

[0067] II. Expression and purification of recombinant protein of Escherichia coli [[ID=3२]]

[0068] 1. Sequence analysis of the gene and selection of the antigen sequence

[0069] Based on the protein identification results in the honey of Leucosceptrum canum, the full length of the Leucosceptrum canum Ycf1 protein is 1839 amino acids. Bioinformatics analysis shows that this sequence contains a typical transmembrane domain. If full-length expression is carried out, it is expected to be toxic due to the insertion of this domain into the Escherichia coli cell membrane and the expression product will be completely insoluble. Therefore, a comprehensive analysis of the transmembrane region, hydrophilicity and disorderliness of the Ycf1 protein sequence was carried out, and finally, the 255-698 amino acid sequence (SEQ ID NO:1) without the transmembrane region was rationally selected for expression in Escherichia coli to prepare the recombinant Leucosceptrum canum Ycf1 protein.

[0070] 2. Protein preparation and purification

[0071] (1) Construction and low-level expression of recombinant plasmids

[0072] Primers were designed to construct expression vectors for E. coli PET-28a (tag size approximately 4kD) or pet-sumo (18kD) lysing vectors. After construction, the sequencing results were 100% correct and mutation-free. The experiments were then conducted to verify the results. Secretory expression was achieved as much as possible through lysing tags and temperature-induced methods.

[0073] (2) Large-scale expression and protein purification

[0074] To expand the scale, the optimal support was selected. First, affinity chromatography was performed using a metal-chelated nickel column. Then, a high-affinity Ni-NTA purification medium was used to covalently couple the chelating agent (nitroglycerin triacetic acid or NTA) onto an agarose medium (4% crosslinking). Finally, Ni was chelated... 2+ It was prepared by means of NTA. NTA can strongly chelate Ni at four sites. 2+ This reduces Ni during the purification process. 2+ It leaked into the protein sample.

[0075] 3. Protein purity detection

[0076] (1) Construction and low-level expression of recombinant plasmids

[0077] The eluted protein solution was placed in a dialysis belt and dialyzed with 1×PBS at 4°C (with two buffer changes). The concentration was over 80% after ultrafiltration at 4°C.

[0078] (2) Large-scale expression and protein purification

[0079] After expressing E. coli in large quantities, the supernatant and precipitated proteins from the lysed cells were analyzed by SDS-PAGE. The results are shown in the figure. Figure 1 The recombinant target protein was found in inclusion body proteins. After recombinant expression in E. coli, the culture volume was increased, and the recombinant protein was purified. The protein concentration was measured to be 1 mg / mL, and the purity was 85%.

[0080] Example 2: Antibody pairing screening and determination of the optimal pairing combination

[0081] 1. Immunization: Balb / c mice were immunized with purified Ycf1 recombinant protein as an immunogen.

[0082] 2. Cell fusion and screening: Spleen cells from immunized mice were fused with myeloma cells SP2 / 0, and hybridoma cells were screened using HAT selection medium.

[0083] 3. Subcloning and screening: Positive hybridoma cells were subcloned at least twice using the limiting dilution method, and then screened using indirect ELISA to obtain monoclonal cell lines that could stably secrete anti-Ycf1 protein.

[0084] 4. Specificity identification: Through cross-reactivity experiments, Ycf1 specific cell lines that do not react with proteins from other nectar-producing plants were screened out.

[0085] 5. Antibody Pairing: Paired antibodies were screened from specific cell lines to ultimately determine the antibody pairs secreted by monoclonal cell lines A11 and C9 for constructing the detection product. The optimal antibody pairing was obtained through the following method:

[0086] A series of anti-Ycf1 protein monoclonal antibodies were screened using a double-antibody sandwich method, and the detection efficacy of different antibody pairs when used in combination was evaluated to determine the optimal antibody pair for high-sensitivity and high-specificity immunoassay.

[0087] 1. Antibodies: The purified mouse monoclonal antibodies prepared in this invention include A11, C9, B3, B12, etc.

[0088] 2. Antigens: Recombinant Ycf1 protein standards at various concentrations, as well as a variety of real honey samples (including other honeys and rice honey).

[0089] 3. Methods: A checkerboard method was used for double-antibody sandwich ELISA. The specific procedure is as follows: Different capture antibodies were coated onto the ELISA plate at a concentration of 2 μg / mL; after blocking, serially diluted standards or honey samples to be tested at a 1:5 dilution were added; after washing, biotin-labeled detection antibody at a working dilution of 1:2000 was added; after incubation with streptavidin-HRP, TMB substrate was used for color development, and the OD450 value was measured.

[0090] Table 2 clearly shows that there are significant differences in the detection performance of different antibody combinations. Among them, the A11+C9 combination exhibits the best performance.

[0091] Table 2 Antibody pairing screening and pairing comparison

[0092]

[0093] Example 3: A colloidal gold test strip for detecting the authenticity of honey in rice flower.

[0094] This embodiment provides a test strip, mainly composed of an absorbent pad, a nitrocellulose membrane, a gold-labeled pad, and a sample pad. The nitrocellulose membrane has a T-line for detecting honey from *Honey styrax* and a C-line for controlling honey, spaced 5 mm apart. The absorbent pad, nitrocellulose membrane, gold-labeled pad, and sample pad are sequentially adhered to a backing plate. An example of the test strip is shown below. Figure 2 .

[0095] Based on the Ycf1-specific paired antibody obtained above, the present invention constructs a colloidal gold immunochromatographic test strip for detecting rice flower honey as follows:

[0096] 1. Preparation of colloidal gold and preparation of gold-labeled antibody complexes

[0097] Colloidal gold solution was prepared using the trisodium citrate reduction method. The pH of the colloidal gold solution was adjusted to the optimal range, and then purified detection antibody (C9) was added under continuous stirring to stably bind to the colloidal gold particles. Bovine serum albumin (BSA) solution was added for blocking to stabilize the gold-labeled antibody complex and reduce non-specific adsorption. The resulting gold-labeled antibody complex was purified by high-speed centrifugation and resuspended in a buffer containing stabilizer to prepare the working solution for the gold-labeled pad.

[0098] 2. Assembly of the test strips

[0099] The test strip consists of a sample pad, a gold label pad, a nitrocellulose (NC) membrane, and an absorbent pad that are sequentially overlapped on a PVC backing.

[0100] Gold-labeled pad: Prepared by uniformly spraying the prepared gold-labeled antibody working solution onto a glass fiber membrane and then drying it.

[0101] Treatment of the reactive membrane (NC membrane):

[0102] Detection line (T line): Capture antibody (A11) is streaked onto the NC membrane at a concentration of 1.5 mg / mL.

[0103] Control line (C line): Goat anti-mouse IgG antibody was streaked onto the NC membrane at a concentration of 1.0 mg / mL.

[0104] Sample pad: A glass fiber membrane is used and treated with a solution containing pH buffer salts, surfactants and blocking agents to optimize sample flowability and reduce background interference.

[0105] Assembly: Paste the prepared sample pad, gold label pad, NC membrane and absorbent pad onto the PVC backing in sequence, and cut them into test strips of appropriate width for use and storage.

[0106] 3. Result Determination

[0107] Positive: Both the T and C lines are red, indicating that the sample contains rice flower honey.

[0108] Negative: Only the C line is red, and the T line is not colored, indicating that no honey component of rice flower was detected in the sample.

[0109] Invalid: If the C line does not develop color, regardless of whether the T line develops color, the test strip is invalid.

[0110] Example 4: Specificity determination of colloidal gold immunochromatographic test strip for distinguishing honey from *Rhizophora stylosa*

[0111] Twenty-five samples of honey from *Apis macrantha*, 20 samples of honey from *Apis rubra*, and 20 samples of honey from *Apis rubra* were obtained from the market, as shown in Table 3. The test strips prepared in Example 2 were used for testing, and the results were observed after 15 minutes. The test results are as follows: Figure 3 As shown in the figure. The results showed that all samples showed a distinct T line at 5 min; only the honey in the rice flower showed a distinct C line at 15 min, demonstrating that this test strip has good specificity.

[0112] Table 3 Honey Sample Information

[0113]

[0114] Example 5: Repeatability determination of colloidal gold immunochromatographic test strips for differentiating honey from *Rhizophora stylosa*.

[0115] Ten honey samples from rice flower buds were randomly selected at different times according to the method in Example 3 for repeated experiments, with a two-day interval between the two batches of test strips. The test results are as follows: Figure 4 As shown in the figure. The results showed no significant difference between the two batches of test strips.

[0116] Example 6: Sensitivity test of colloidal gold immunochromatographic test strip for distinguishing honey from *Rhizophora stylosa*.

[0117] Following the method in Example 4, ten honey samples from rice flower clusters were randomly selected. The honey samples were diluted with distilled water to eight concentrations (50%, 45%, 40%, 35%, 30%, 25%, 20%, and 15% by mass) for sensitivity testing. The lowest dilution concentration at which a positive result was observed was taken as the lower limit of detection (LOD) for the test strip. The test results are as follows: Figure 5 As shown. The limit of detection can distinguish honey diluted in rice flower at a concentration of up to 25%. Results must be interpreted within 15–45 minutes; results after 45 minutes are invalid.

[0118] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of Ycf1 protein or its truncated form: 1) Used for the identification of rice flower honey; 2) As a characteristic marker of rice flower honey; 3) Used to construct the fingerprint spectrum of rice flower honey; The amino acid sequence of the Ycf1 protein is shown in SEQ ID NO:2; in, The truncated version of the Ycf1 protein is the amino acid sequence from position 255 to 698 of the Ycf1 protein.

2. A method for authenticating rice flower honey, characterized in that, If the components in the honey to be tested are detected, and Ycf1 protein or its truncated form is found, then the honey to be tested is either rice flower honey or adulterated with rice flower honey. The Ycf1 protein or its truncated form is the same as the Ycf1 protein or its truncated form described in claim 1.

3. A monoclonal antibody against Ycf1 protein, characterized in that, The monoclonal antibody is secreted by hybridoma cell line A11 with accession number CGMCC No. 46751 or hybridoma cell line C9 with accession number CGMCC No. 46752.

4. A composition, characterized in that, The composition comprises monoclonal antibodies secreted by hybridoma cell line A11 with accession number CGMCC No. 46751 and monoclonal antibodies secreted by hybridoma cell line C9 with accession number CGMCC No. 46752.

5. Any of the following applications of the anti-Ycf1 protein monoclonal antibody of claim 3 or the composition of claim 4: (i) Used to detect Ycf1 protein or its truncated form in samples; (ii) Prepare reagents or test strips for detecting Ycf1 protein or its truncated form; (iii) Prepare a kit for detecting Ycf1 protein or its truncated form.

6. A colloidal gold immunochromatographic test strip for rice honey, comprising a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad stacked sequentially, characterized in that... The gold-labeled pad is coated with a gold-labeled antibody formed by the combination of a first antibody and colloidal gold; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with a second antibody, and the control line is coated with goat anti-mouse IgG. Wherein, the first antibody is a monoclonal antibody secreted by hybridoma cell line A11 with accession number CGMCC No. 46751, and the second antibody is a monoclonal antibody secreted by hybridoma cell line C9 with accession number CGMCC No. 46752; or, The first antibody is a monoclonal antibody secreted by hybridoma cell line C9 with accession number CGMCC No.46752, and the second antibody is a monoclonal antibody secreted by hybridoma cell line A11 with accession number CGMCC No.46751.

7. The test strip according to claim 6, characterized in that, The sample pad is a glass fiber membrane.

8. The method for preparing the test strip according to claim 6 or 7, characterized in that, Includes the following steps: (1) Prepare colloidal gold solution; (2) Add the purified first antibody to the colloidal gold solution and stir to make the first antibody stably bind to the colloidal gold particles; (3) Add bovine serum albumin solution for blocking, centrifuge at high speed for purification, resuspend in buffer containing stabilizer to prepare gold pad working solution; (4) Spray the gold-labeled pad working solution evenly onto the glass fiber membrane, dry it, and prepare the gold-labeled pad; (5) The second antibody is streaked onto a nitrocellulose membrane at a certain concentration to form a detection line; (6) Sheep anti-mouse IgG antibody was streaked onto a nitrocellulose membrane at a certain concentration to form a quality control line; (7) Stack the sample pad, gold label pad, nitrocellulose membrane and absorbent pad on the PVC backing in sequence, and cut them into test strips of appropriate width.

9. The use of the test strip according to claim 6 or 7 in the detection of rice flower honey.

10. A method for detecting rice flower honey using the test strip according to claim 6 or 7, characterized in that, The method includes: diluting the honey sample to be tested with water, inserting a test strip, allowing the diluted honey sample to saturate the sample pad, and reading the result after a period of time; The results are judged as follows: when the control line is red, the result is valid; when the test line shows color, the honey sample is determined to be honey from rice flower or honey from rice flower that has been mixed in; when the control line does not show color, the result is invalid.

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