CN115236336A

CN115236336A - Preparation and application of colloidal gold test strip for simultaneously detecting sources of honey and Italian honey bee species

Info

Publication number: CN115236336A
Application number: CN202210811978.0A
Authority: CN
Inventors: 胡福良; 张言政; 陈伊凡; 张国志; 李珊珊; 郑火青; 张翠平
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2022-10-25

Abstract

The invention provides a preparation method and application of a colloidal gold test strip for simultaneously detecting sources of honey and honey Italian bees. The test strip consists of an absorption pad, a nitrocellulose membrane, a gold-labeled pad, a sample pad and a polyvinyl chloride base plate, wherein the nitrocellulose membrane is provided with a T1 Italian honey detection line, a T2 Chinese honey detection line and a C quality control line. The preparation method comprises the following steps: (1) Preparing and purifying a specific Apis mellifera MRJP2 monoclonal antibody and a Apis cerana MRJP2 monoclonal antibody; (2) preparing a colloidal gold solution; (3) A quality control line (C line), an Italian honey detection line (T1 line) and a medium honey detection line (T2 line) are coated on the nitrocellulose membrane; and (4) assembling the test strip. The colloidal gold test strip disclosed by the invention can be used for simultaneously detecting the source authenticity of the honey and the Italian honey on one test strip, is suitable for efficiently and quickly detecting the Italian honey mixed in the honey, and can also be used for identifying pure syrup type fake honey.

Description

Preparation and application of colloidal gold test strip for simultaneously detecting sources of medium-honey and Italian honey bee species

Technical Field

The invention belongs to the field of bee product detection, and relates to a colloidal gold test strip for simultaneously detecting sources of honey and honey Italian bee species, and preparation and application thereof.

Technical Field

The Chinese bee is also called as native bee, which is a native bee variety in China. Chinese honey is a unique honey variety in China, is popular with China, but has low yield, and the retail price of Chinese honey is generally 160-400 yuan/kg, which is five to ten times that of Chinese honey. Driven by interests, phenomena of honey impersonation or honey incorporation in the honey on the market sometimes occur, and consumers and practitioners of the honey suffer losses.

The honey contains about 1% of protein, and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis shows that the honey protein is mainly royal jelly major protein MRJPS secreted by bees, wherein the highest MRJP1-3 content is an inherent and stably existing component in the honey and cannot be replaced by other exogenous substances mixed into the honey. According to the immune characteristics, MRJPs are used as endogenous markers for bee species source detection of honey, which is a feasible idea. MRJPs of the Chinese bees and the Italian bees have bee species difference, which provides possibility for preparing specific antibodies of the two. As can be seen from the homology analysis of Membristus and Apis MRJP1-3, the homology of both MRJP1 is 91.22%, the homology of MRJP2 is 83.76%, the homology of MRJP3 is 77.83%, the homology of MRJP1 of Membristus and Apis is too high, and MRJP3 is highly polymorphic, so MRJP2 is preferably selected as the target antigen for antibody production.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip for simultaneously detecting bee species sources of medium honey and honey Italian honey, which can simultaneously display the bee species source information of the medium honey and honey Italian honey on one test strip. The test strip contains a specific Italian bee MRJP2 monoclonal antibody and a specific Meretrix bee MRJP2 monoclonal antibody, the specific Italian bee MRJP2 monoclonal antibody is obtained by secretion of a 35E2 cell strain, and the preservation number of the 35E2 cell strain is as follows: CCTCC NO: C2022208, classification name: hybridoma cell line 35E2, depository: china center for type culture Collection, collection Address: wuhan university, storage day: 7/6/2022; the specific Apis cerana MRJP2 monoclonal antibody is obtained by secreting 30G10 cell strain, and the preservation number of the 30G10 cell strain is as follows: CCTCC NO: C2022209, classification name: hybridoma cell line 30G10, depository unit: china center for type culture Collection, collection address: wuhan university, storage day: 7/6/2022.

The test paper strip mainly comprises absorption pad, nitrocellulose membrane, gold mark pad, sample pad, be equipped with honey detection line and C matter accuse line in T1 Italian honey detection line, T2 on the nitrocellulose membrane, C line, T1 line and T2 line respectively are 5mm apart, absorption pad, nitrocellulose membrane, gold mark pad, sample pad, paste in proper order on the plastics welt, pack into plastic casing.

The invention also aims to provide a preparation method of the test strip, which is realized by the following technical scheme:

1. separation and purification of antigen

(1) Dialyzing and purifying royal jelly of Apis cerana Fabricius and Apis mellifera by using a 14kDa dialysis bag, centrifuging 10000g of the dialyzed royal jelly solution for 30min, and filtering the supernatant by a 0.22 mu m filter membrane to obtain filtrate, namely the crude extract of the main protein (MRJPS) of royal jelly;

(2) Purifying antigen protein by column chromatography, selecting QSepharose F.F. (20 mL) as a glass column number, identifying each eluted component by electrophoresis, and selecting Apis cerana MRJP2 and Apis mellifera MRJP2 as antigens for subsequent immunization.

2. Immunization of mice

Purified Chinese bee MRJP2 and Apis mellifera MRJP2 are used as antigens for immunization, female BALB/c mice of 8 weeks old are selected as immunized animals, serum 7 days after the third immunization is taken, the serum antibody level is measured by an indirect ELISA method, and if the serum reaches a fusion standard (the serum is diluted by 10000 times, and OD450 is more than 1), the mice with high titer are selected to be boosted for one time in 3 days before the fusion.

3. Fusion of mouse splenocytes with myeloma cells

According to the monoclonal preparation process, eight Italian bee MRJP2 fusion cells are obtained according to the specificity of Italian honey, wherein two 33C6 and 24D6 are subcloned and turned to be negative, and one cloning well with a good cell state is selected from the remaining six fusion cells 3B2, 31E2, 27D12, 1C1, 35C4 and 35E2 of Italian bee MRJP2 for secondary subcloning.

According to the specificity of the medium honey, six Italian bee MRJP2 fusion cells are obtained, wherein 9B1 is not positive by an Elisa double-antibody sandwich method, 22F12 turns negative, and one cloning well with a better cell state is selected from the fusion cells 13A5, 30G10, 25D10 and 11E7 of the rest four Italian bee MRJP2 respectively for secondary subcloning.

The Elisa double-antibody sandwich test result shows that the Italian bee MRJP2 screening clone cells all have sandwich effect, stable strains are preserved, inoculated mice are used for preparing ascites, the antibody titer of the Mesona bee MRJP2 cell strain is 25D10, 30G10 >1E7 >13A5, and when the antigen concentration is high (1 ug/ml), the Mesona bee MRJP2 cell strain and the Italian bee MRJP2 have cross reaction. And (3) performing expanded culture and cryopreservation on the Chinese bee MRJP2 stable cell strain, and inoculating a mouse to prepare ascites.

4. Preparation of rabbit anti-Mesona protein polyclonal antiserum

Selecting a new Zealand rabbit weighing about 2kg, taking purified Mesona MRJP2 and Italian bee MRJP2 proteins as antigens for immunization, immunizing for 3 times totally, wherein the immunizing dose is 1 mg/rabbit, collecting blood from ear marginal veins after 7 days of three-immunization, detecting the serum antibody level by ELISA, taking blood from heart after the titer meets the requirement, separating serum, purifying and dialyzing through an antigen affinity column to obtain a polyclonal antibody capable of simultaneously identifying the Mesona MRJP2 and the Italian bee MRJP2, and taking the polyclonal antibody as a coating antibody of a colloidal gold test strip.

5. Pairing and marking of apis mellifera and apis mellifera honey monoclonal antibodies

The specific Apis mellifera MRJP2 monoclonal antibody selects a 35E2 cell strain (preservation number: CCTCC NO: C2022208), and the monoclonal antibody secreted by the 35E2 cell strain has strong specificity and does not have cross reaction with the Apis mellifera MRJP2. The Apis cerana MRJP2 monoclonal antibody selects 30G10 cell strain (preservation number: CCTCC NO: C2022209), and the monoclonal antibody secreted by the 30G10 cell strain has cross reaction with Apis cerana MRJP2.

6. Preparation of colloidal gold test strip

(1) Preparing a colloidal gold solution: preparing a chloroauric acid aqueous solution into a colloidal gold solution containing colloidal gold particles with the particle size of about 20 nm;

(2) Colloidal gold-labeled antibody: adding polyclonal antibodies capable of simultaneously identifying Mesema MRJP2 and Italian MRJP2 into colloidal gold solution to prepare immune colloidal gold, wherein the optimal pH of the colloidal gold antibody is 8.5; attaching immune colloidal gold on non-woven fabric to form a gold label pad;

(3) Coating a C line, a T1 line and a T2 line on a nitrocellulose membrane, wherein the C line is a quality control line, a commercial goat anti-mouse lgG is coated, the T1 line is an Italian honey detection line, the T2 line is a Membristein honey detection line, and the T1 line and the T2 line are coated with rabbit polyclonal antibodies capable of identifying Membristian MRJP2 and Italian MRJP2 simultaneously. The detection antibody of the T1 line is a specific Apis mellifera MRJP2 monoclonal antibody, and the detection antibody of the T2 line is a Apis cerana MRJP2 monoclonal antibody. Dotting the cellulose nitrate membrane by using a membrane scribing machine to obtain a C line, a T1 line and a T2 line, wherein the C line, the T1 line and the T2 line are separated by 5mm; drying at room temperature to obtain a coated nitrocellulose membrane;

(4) Assembling the colloidal gold immunochromatographic rapid detection test strip: sequentially sticking an absorption pad, a nitrocellulose membrane, a gold label pad and a sample pad on a polyvinyl chloride (PVC) bottom plate from top to bottom, cutting into strips with the width of 2-3mm, and filling the strips into a plastic shell to obtain the rapid detection test strip; drying and storing at 4 ℃ in dark.

The invention aims to provide application of the colloidal gold test strip in detection of Chinese bee honey and Italian bee honey, and the application comprises detection of source authenticity of Chinese bee honey and Italian bee honey and detection of adulterated Chinese bee honey

The application of the invention is realized by the following steps:

(1) taking out the test strip, and returning to room temperature;

(2) preparation of honey sample detection solution: mixing a honey sample with pure water in a mass ratio of 1:10, diluting and uniformly mixing;

(3) the test paper strip is horizontally placed, 2 drops of diluted honey sample are added on the sample pad, and the result is read after 5-10 minutes;

(4) and (5) judging a result: the results are valid when the C line is displayed as a red line. When the T1 line and the T2 line are developed, the result is apis mellifera honey or apis mellifera honey is mixed, namely the sample to be detected contains apis mellifera MRJP2; when the T1 line is not developed and the T2 line is developed, the result is medium honey, and the sample to be detected only contains the MRJP2 of the medium bee and does not contain the MRJP2 of the Apis cerana; neither the T1 nor T2 lines developed, resulting in syrup, i.e., the sample contained neither apis cerana MRJP2 nor apis cerana MRJP2. When the C line did not develop color, the results were not valid.

The colloidal gold test strip disclosed by the invention is suitable for quickly and accurately detecting the source authenticity of the Chinese honey and the Italian honey bee species, can simultaneously detect the source authenticity of the Chinese honey and the Italian honey bee species on one test strip, is suitable for efficiently and conveniently detecting the mixture of the Chinese honey and the Italian honey in a certain proportion, and can also be used for identifying the false honey of a pure syrup type without main proteins of the royal jelly.

The colloidal gold immunochromatographic test strip technology is a novel colloidal gold labeled immunochromatographic technology, has the characteristics of visualization, strong specificity, rapidness, convenience and the like, can be used for indirect qualitative or semi-quantitative detection, has low requirements on detection personnel, and is very suitable for on-site rapid detection of medium honey. In order to meet the requirements of quick and simple detection of honey in the bee product industry, consumers and medium honey, the invention provides a colloidal gold test strip for quickly detecting authenticity of medium honey and honey Italian bee species, and a preparation method and application thereof. The Apis mellifera MRJP2 monoclonal antibody and the Apis cerana MRJP2 monoclonal antibody are prepared by a monoclonal antibody preparation technology, the Apis mellifera MRJP2 monoclonal antibody can only specifically identify Apis mellifera MRJP2 protein, and can not identify Apis cerana MRJP2 protein. The Apis mellifera MRJP2 monoclonal antibody has the advantages of high sensitivity, strong specificity and the like, and has no cross reaction with other proteins related to the royal jelly major protein family.

The invention has the beneficial effects that: the colloidal gold test strip for detecting the authenticity of the Chinese honey and the Italian honey bee species has the advantages of convenience and quickness in operation, short detection time and the like, is suitable for identifying the authenticity of the Chinese honey bee species, can accurately and quickly detect the addition of more than 20% of Italian honey in the Chinese honey, and can also be used for identifying pure syrup honey.

Drawings

FIG. 1 is a schematic view of a test strip.

FIG. 2 is an SDS-PAGE image of purified natural Apis mellifera MRJP2 protein and Apis cerana MRJP2 protein.

FIG. 3 shows the specific identification of two Apis cerana MRJP2 monoclonal antibodies.

FIG. 4 shows the actual detection effect of the authenticity colloidal gold test paper for Chinese honey and Italian honey bee species.

Detailed Description

The present invention will be further described with reference to the following examples, which are provided for illustration of the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional in the art, and all reagents used are analytically pure.

Example 1: colloidal gold test strip for simultaneously detecting sources of honey and Italian honey bee species

The embodiment provides a test paper strip, mainly comprises absorption pad, nitrocellulose membrane, gold mark pad, sample pad, be equipped with honey detection line and C matter accuse line in T1 Italian honey detection line, T2 on the nitrocellulose membrane, C line, T1 line and T2 line respectively are 5mm apart from, absorption pad, nitrocellulose membrane, gold mark pad, sample pad, paste in proper order on the plastics welt, pack into plastic casing, the test paper strip example is shown in figure 1. The test strip contains a specific Apis mellifera MRJP2 monoclonal antibody and a specific Apis cerana MRJP2 monoclonal antibody, the specific Apis mellifera MRJP2 monoclonal antibody is obtained by secreting a 35E2 cell strain, and the preservation number of the 35E2 cell strain is as follows: CCTCC NO: C2022208, the specific Chinese bee MRJP2 monoclonal antibody is obtained by the secretion of 30G10 cell strain, the preservation number of the 30G10 cell strain is as follows: CCTCC NO: C2022209. The 35E2 cell line and the 30G10 cell line are preserved by the China center for type culture Collection, and the preservation date is as follows: 7/6/2022.

Example 2: separation and purification of natural apis cerana MRJP2 and apis mellifera MRJP2 antigens

According to the homology analysis of MRJP1-3 of Chinese bees and apis mellifera, the homology of MRJP1 of the two is 91.22%, the homology of MRJP2 is 83.76%, the homology of MRJP3 is 77.83%, the homology of MRJP1 of Chinese bees and apis mellifera is too high, and MRJP3 has high polymorphism, so that MRJP2 is preferably selected as a target antigen for antibody preparation, and the MRJP2 antigen is obtained and directly separated and purified from royal jelly.

1. Sample processing

1) Weighing fresh Lac Regis Apis 3g, dissolving in 30mL pure water, and stirring gently with magnetic stirrer at 4 deg.C for 3 hr. Placing the solution in a 50mL centrifuge tube, centrifuging for 30min at 10000g, and loading the supernatant into a 14kDa dialysis bag;

2) Placing the dialysis bag in 3000mL of pure water, dialyzing at 4 ℃ for 24h, and changing water once within 8 h;

3) Centrifuging 10000g of the dialyzed royal jelly solution for 30min, and filtering the supernatant through a 0.22 mu m filter membrane to obtain a filtrate, namely the royal jelly major protein crude extract.

2. Antigen purification

The purification of the antigen protein is carried out by a column chromatography method, which comprises the following specific operations:

1) Chromatography column and eluent selection

Glass chromatography column (20 mL) qsepharose f.

Eluent A:0.01mol/L PB buffer, pH =7.6

Eluent B:0.01mol/L PB buffer, pH =7.6, containing 5 concentration gradients of NaCl, 0.05mol/L, 0.1mol/L,0.2mol/L,0.3mol/L,0.4mol/L, each for 2 column volumes).

2) Column mounting

One column was filled with about 10mL of the eluent a, and qsepharose f.f. suspended in an appropriate amount of the eluent a was poured into the column while stirring, and allowed to settle naturally until all was added. The column was equilibrated with 5 column volumes of eluent A at a flow rate of 1.0 mL/min.

3) Sample loading

After the above equilibration process, after the liquid level was about 1cm from the resin settling surface, 20mL of sample was carefully added along the tube wall using a dropper, and the effluent was collected.

4) Elution is carried out

Eluting 2 column volumes respectively with 5 gradients in eluent A and eluent B, and collecting respectively.

5) The eluted fractions were analyzed by SDS-PAGE and the gel strips were destained by Coomassie blue staining to give clear bands for each fraction (FIG. 2). According to molecular weight, judging the Mesema MRJP2 and the Apis mellifera MRJP2, and carrying out electrophoretic display: the protein eluted by NaCl with the concentration of 0.4M has the purity of more than 90 percent and can be used for animal immunization.

Example 3: apis mellifera MRJP2 and Apis cerana MRJP2 monoclonal antibody preparation

1. Immunization of mice

Purified Apis cerana MRJP2 and Apis mellifera MRJP2 are used as antigens for immunization, female BALB/c mice of 8 weeks of age are selected as the immunized animals, and the detailed immunization procedures are shown in Table 1. Taking serum 7d after the third immunization, measuring the serum antibody level by an indirect ELISA method, and selecting mice with high titer to perform boosting immunization once 3d before fusion if the serum reaches the fusion standard (the serum is diluted by 10000 times, and OD450 is more than 1).

TABLE 1 immunization procedure

The serum titers of the immunized mice were determined, and the results are shown in tables 2, 3, 4 and 5.

Table 2 o wasp MRJP2 mice serum titer detection OD450 readings after three immunizations

	Left front	Right front	Left back	Right back	Is free of	Blood of empty white mouse
							1/1000	1.8694	2.0471	2.0458	2.0967	2.1018	0.0898
1/3000	1.4014	1.8181	1.8296	1.8519	1.9306	0.0667
							1/9000	0.9014	1.5301	1.5378	1.4195	1.5494	0.0606
1/27000	0.4656	1.065	0.9362	0.8718	0.94	0.0436
							1/81000	0.293	0.6601	0.5606	0.4816	0.5749	0.1291
1/243000	0.1039	0.267	0.2165	0.192	0.2215	0.0458
							1/792000	0.088	0.1461	0.1302	0.1149	0.1282	0.0663
Anti-rarefaction	0.0635	0.058	0.0562	0.0584	0.0546	0.0572

Table 3 cross detection of mouse serum OD450 readings after three immunizations of apis mellifera MRJP2

	Left front	Right front	Left back	Right back	Is free of	Blood of empty rat
							1/1000	1.8628	1.9621	2.0747	1.9863	1.7003	0.0794
1/3000	1.7014	1.7258	1.8492	1.6282	1.3783	0.0699
							1/9000	1.3834	1.3099	1.4261	1.0769	0.8313	0.0587
1/27000	0.8326	0.79	0.8991	0.5819	0.3955	0.0605
							1/81000	0.5326	0.4361	0.5269	0.3435	0.2582	0.1188
1/243000	0.2204	0.1654	0.1994	0.1195	0.0941	0.0368
							1/792000	0.1216	0.0942	0.1229	0.0941	0.0834	0.0618
Resist dilution	0.061	0.0555	0.0698	0.0604	0.072	0.0575

The result shows that the serum titers of 5 mice after three times of immunization by the Apis mellifera MRJP2 are similar and can reach 1.

TABLE 4 OD450 readings for serum titer detection in mice after three immunizations of Apis cerana MRJP2

	Left front	Right front	Left back	Right back	Is composed of	Blood of empty rat
							1/1000	2.1464	1.874	2.1183	2.0622	2.0744	0.0818
1/3000	1.9718	1.8328	1.9947	1.8742	1.918	0.0673
							1/9000	1.7621	1.8277	1.9515	1.7352	1.7589	0.0533
1/27000	1.3704	1.5816	1.7132	1.4238	1.3888	0.0449
							1/81000	0.8766	1.1562	1.3435	1.0273	0.9834	0.1145
1/243000	0.3702	0.5633	0.7432	0.492	0.4952	0.0391
							1/792000	0.1731	0.2751	0.3757	0.2418	0.2488	0.0609
Resist dilution	0.0647	0.0668	0.0607	0.0532	0.054	0.0559

TABLE 5 OD450 readings from mouse serum cross-detection after three immunizations of bee MRJP2

The result shows that the serum titers of 5 mice after three immunizations of the Chinese bee MRJP2 are similar and can reach 1.

2. Fusion of mouse splenocytes with myeloma cells

2.1 preparation of feeder cells

Taking 6 12-24h before fusionBa1b/c mice at-8 weeks of age, were bled from the orbit and mouse negative sera were collected for use. After the cervical vertebra dies at the dislocation, the cervical vertebra is soaked in 75% alcohol solution for 5min. Separating abdominal wall and peritoneum, injecting appropriate amount of serum-free 1640 culture solution into mouse abdominal cavity via peritoneum, massaging or tapping abdominal wall for 2min to form cell suspension. Extracting the suspension, immediately transferring the suspension into a pre-cooled 15mL centrifuge tube, preventing macrophage from adhering to the wall by ice bath, centrifuging at 1500g for 5min, and discarding the supernatant. After cell counting (total number of cells should reach 4X 10) ⁶ Individual cells) were suspended in HAT medium, and the cell concentration was adjusted to 1-5X 10 ⁵ and/mL. Washing with a pipette, mixing, adding into 96-well plate, 100 μ L/well, adding into the mixture at 37 deg.C, 5% CO ₂ Culturing in an incubator.

2.2 preparation of myeloma cells

The myeloma cells are recovered two weeks before the fusion, subcultured every 2-3 days, and can be used for the fusion when the cells grow vigorously and have good shapes (uniform size, round and transparent). One day before cell fusion, cell concentration was adjusted to 2X 10 with fresh medium ⁵ and/mL, the next day is generally cells in a logarithmic growth phase, and the fusion success rate is higher. Myeloma cells are collected in a 50mL centrifuge tube, centrifuged for 10min at 1200g, the supernatant is discarded, the packed cells are resuspended in a basal medium, centrifuged again under the same conditions, and the supernatant is discarded. Resuspending with appropriate amount of basal medium (cell density 1-2X 10) ⁶ Individual cells/mL, 50-100 per large compartment).

Cell counting: and (4) sucking the cell suspension by using a micropipette, adding one drop of the cell suspension to the side of a cover glass of the blood counting plate, diffusing the cell suspension into a counting chamber, and counting the number of cells in the square grid under a 10-time objective lens. Sucking 1-2X 10 ⁷ And (4) preparing each cell for later use.

2.3 mouse spleen cell preparation

And taking the mice after the boosting immunization, sampling blood in the orbit, and separating serum to be used as a positive control for later use. Soaking the mouse in 75% alcohol for 5min, opening the abdominal cavity of the mouse, taking out the spleen, stripping the fascia, grinding the mouse spleen on a 200 mesh screen, washing the mesh screen with 10mL of 3% FBS1640, and collecting the spleen cell suspension. 1200g was centrifuged for 10min, the spleen cell pellet was resuspended in 10mL 3% FBS1640, and the cells were counted after mixing.

2.4 cell fusion

Immune spleen cells and SP2/0 myeloma cells were mixed well at a ratio of 5. Flick the bottom of the centrifuge tube gently to loosen the cell pellet, add PEG dropwise within 1min, and mix gently. Adding 10mL of stop solution (slowly and quickly) in a 37 ℃ water bath for 1min, standing for 5min, sliding along the tube wall, stirring gently, cutting into pieces which can not be blown forcefully to prevent the fused cells from dispersing, and standing for 5min. After centrifugation at 1200g for 5min, the supernatant was discarded and suspended in 25mL HAT medium, and HAT medium containing fused cells was inoculated into a 96-well plate (containing 150. Mu.L of feeder cells) and cultured in a cell culture chamber.

2.5 screening of Positive hybridoma cells

The time and number of the clone wells were recorded and the culture medium was replaced with HT 1640 after 7d of fusion. When the hybridoma colony in the culture well is expanded to 1/4-1/2 of the well area, the specific antibody is detected by indirect ELISA method. And subcloning the screened positive cloning wells with high OD450 nm values.

2.6 subcloning of Positive hybridoma cells

Feeder cells were prepared as described in 2.1 the day before subcloning by limiting dilution. And (4) blowing and beating the cells in the positive holes, transferring the cells to a 1mLHT selection culture medium, mixing the cells uniformly, and counting the cells. Taking 150-200 cells in 10mLHT selection medium, adding 100 μ L of each well into the paved feeder cell culture plate, observing and recording the growth condition of the hybridoma cells. And transferring the remaining cells in the positive holes to a 24-pore plate for amplification culture, and freezing and storing in time. When the hybridoma colony in the culture well is expanded to 1/4-1/2 of the well area, the detection is carried out by indirect ELISA. And continuously cloning the screened positive cloning wells with high OD450 nm values. At least 3-5 times of cloning culture work is generally carried out until 10-20 monoclonal strains selected in the last time are detected as all positive clones, namely 100% positive porosity.

TABLE 6 ELISA Sandwich method for detecting OD450 readings of hybridoma supernatants after first subcloning of Apis mellifera MRJP2

The results showed that one well of 3B2, 31E2, 27D12, 1C1, 35C4, and 35E2 was selected for secondary subcloning.

Table 7 OD450 readings from ELISA sandwich rechecking of hybridoma supernatants after first subcloning of bee MRJP2

The results showed that the 9B1 sandwich method was not positive and 22F12 turned negative. Therefore, one well of 13A5, 30G10, 25D10, and 11E7 was selected for secondary subcloning.

According to the ELISA cross detection OD450 reading of hybridoma supernatant after the second subcloning of Apis mellifera MRJP2, the indirect method detection result is as follows: 31E2, 35e2>, 35c4, 27d12>, 3b2, 1C1. The detection result of the sandwich method shows that the cloned cells screened by the Italian bee MRJP2 have sandwich effect, the stable strains are preserved, and the mice are inoculated to prepare ascites.

According to the detection result of the hybridoma supernatant ELISA cross detection OD450 reading indirect method after the second subcloning of the Chinese bee MRJP 2: 25D10, 30G10 >. And (3) detection results of a sandwich method: 25D10, 30G10 >. And (3) integrating detection results of an indirect method and a sandwich method, performing expanded culture and cryopreservation on the MR2 stable cell strain, and inoculating a mouse to prepare ascites.

2.7 identification of monoclonal antibodies

Specificity identification (Indirect ELISA method)

1) Coating the apis mellifera protein: coating buffer diluted Apis protein to 2. Mu.g/mL, 100. Mu.L/well. Overnight at 4 ℃;

2) Coating Chinese bee protein by the same method;

3) 5% skimmed milk powder sealed, 200. Mu.L/well. Incubating at 37 ℃ for 2h;

4) PBST wash 5 times;

5) And adding the positive monoclonal culture supernatant into the apis mellifera protein coated hole and the apis cerana protein coated hole respectively, wherein the concentration is 100 mu L/hole. Incubating for 1h at 37 ℃;

6) PBST wash 5 times;

7) 1% nonfat milk powder-PBST diluted goat anti-mouse secondary antibody (1: 10000 100 μ L/well;

8) PBST wash 5 times;

9) TMB system color development, 37 degrees C incubation 15min;

10)2M H ₂ SO ₄ terminating the reaction;

11 OD450 nm was measured.

Specificity detection is carried out on 2 hybridoma cell strains (the serial numbers are 35E2 and 35C 4) capable of stably secreting Italian bee protein, and the specificity of the Italian bee honey specific monoclonal antibodies 35E2 and 35C4 is good (figure 3), so that the development of subsequent colloidal gold test paper strips is met.

2.8 preparation of ascites in mice

Injecting 0.3mL special adjuvant into the mouse body one week in advance, recovering the hybridoma cell strain to logarithmic growth phase, with the dosage of 1 × 10 ⁶ One cell per mouse. Generally, the abdominal distension of the mice starts 7-8d after the injection of the hybridoma cells, and ascites is collected at 14 d. Centrifuging to remove impurities, collecting supernatant, and freezing at-80 deg.C.

2.9 ascites purification (antibody purification)

The ascites was removed from the freezer at-80 deg.C, thawed quickly and rewarmed, and filtered through a 0.45 μm filter. 5mL of ascites fluid were centrifuged at 12000g for 10min at room temperature, and the supernatant was transferred to a new 50mL centrifuge tube. Diluted 4-fold with PBS, filtered through a 0.22 μ M filter, and purified on a protein G affinity column. The column was washed with 5 column volumes of PBS, then eluted with 3 column volumes of 0.1m pH 2.7gly-HCl solution, the eluate was collected and neutralized and dialyzed overnight against PBS. Centrifuging at 10000g for 10min, collecting supernatant, filtering with 0.22 μm filter membrane for sterilization, measuring protein content with NanoDrop 2000, packaging, and standing at-80 deg.C. Purity was assessed by SDS-PAGE and antibody titers were determined by indirect ELISA.

TABLE 8 Honey 35E2, 35C4 mouse ascites ELISA assay OD450 readings

The results show that ascites 35E2, 35C4 react well with antigen in an indirect method; the sandwich effect is poor in the detection of the monoclonal antibody-polyclonal antibody sandwich method, and the sandwich effect is only achieved when the antigen concentration is higher

In Table 9 the OD450 readings of ascites ELISA detection of honey 13A5, 30G10 mice

13A5

30G10


	1/1000	1.4257	1.3361
1/3000				1.3865	1.2775
	1/9000	1.3428	1.2503
1/27000				1.3253	1.1342
	1/81000	1.3108	1.0131
1/243000				1.2583	0.8289
	1/79200	1.2574	0.6767
Diluent liquid				0.0614	0.0547

The results show that ascites 13A5, 30G10 reacted well with antigen in the indirect method; the 30G10 sandwich effect is good, the sensitivity is higher, and the 13A5 sandwich effect is not generated in the detection of the monoclonal antibody-polyclonal antibody sandwich method; the results of different capture secondary antibodies of sheep anti-mouse (G-M) and donkey anti-mouse (D-M) are consistent in trend

2.10 monoclonal antibody subtype identification

The supernatant of three hybridoma cells, 35E2, 35C4 and 30G10, was tested by using a mouse monoclonal antibody test kit and reacted with anti-IgG I, igG2a, igG2b, igG3, igA and IgM antibodies, respectively, and OD 450nm >0.8, which was determined to be a positive reaction. The results show that the monoclonal antibodies secreted by the obtained 3 hybridoma cells are all IgG1 type.

Example 4: development of colloidal gold test strip for detecting authenticity of honey and honey Italian bee species

1. Determination of optimal pH value of colloidal gold labeled rabbit anti-apis mellifera polyclonal antibody

Taking a 20nm colloidal gold solution, centrifuging 12000g of polyclonal antibody to be labeled for 20min at 4 ℃, taking supernatant, filtering the supernatant through a 0.45 mu m filter membrane, measuring the antibody concentration by using NanoDrop 2000, and adjusting the antibody concentration to 1mg/mL by using PBS for later use.

Taking 2 columns of enzyme-linked immunosorbent assay plates (8 wells), adding 0.5mL of colloidal gold solution, then adding 50 mu L of polyclonal antibody (1 mg/mL) into the solution with 0.2mol/L K ₂ Co ₃ Adjusting to colloidal gold solution with different pH values, mixing by vortex oscillation, instantly centrifuging, standing for 10min, adding 50 μ L10% NaCl to each tube (except control tube), mixing, standing for 2h, and adding sample information to each well as shown in Table 10.

TABLE 10 determination of the optimum pH of colloidal gold-labeled antibodies

OD 530nm value measured by a microplate reader: and taking 200 mu L of colloidal gold suspension of each tube, measuring the OD 530nm value, and drawing a curve. And (4) judging a result: the pH at which the OD 530nm is at its maximum is the optimum pH. At the moment, the color of the colloidal gold solution in the pipe has no change compared with the original solution and no coagulation appears. The optimal pH of the gold-labeled antibody was determined to be 8.5.

2. Determination of optimum concentration of colloidal gold labeled rabbit anti-apis mellifera polyclonal antibody

And (3) taking 2 columns of enzyme-labeled plates, adding 0.5mL of colloidal gold solution into each enzyme-labeled plate, adjusting the pH value of the label to the optimal label pH value determined in the previous step, adding 50 mu L of polyclonal antibody (1 mg/mL) with different dilution times into each tube, carrying out vortex oscillation, mixing uniformly, and standing for 10min. Add 50. Mu.L of 10% NaCl to each tube (except the control tube), mix, and let stand for 2 hours. As a result, the optimum concentration was determined as the antibody concentration at which the OD 530nm value was the maximum. At the moment, the color of the colloidal gold solution in the pipe has no change compared with the original solution and no coagulation appears. Finally, the concentration of the coated polyclonal antibody and the gold spraying amount are determined to be 8 mu L/cm.

3. Determination of detection line, quality control line coating concentration and gold mark spraying amount

Adjusting the T-coat concentration of the purified monoclonal antibody within the range of 0.1-1.5 mg/mL, adjusting the C-coat concentration within the range of 0.1-1mg/mL, adjusting the gold-spraying amount within the dosage range of 4-10 muL/cm, and taking the conditions when the positive sample C line and the negative sample T line have moderately and uniformly developed bands and the negative sample C line and the negative sample T line do not develop as the final coat concentration and the gold-spraying amount.

4. Development of colloidal gold test strip

4.1 preparing a gold-labeled pad: the search for the gold pad parameters was performed as per table 11.

TABLE 11 gold pad parameters

4.2 spray coating

And determining various parameters of the sprayed film through exploration, wherein the parameters of the sprayed film are shown in a table 12.

TABLE 12 colloidal gold test strip spray parameters

Position of	Name (R)	Initially (mg/ml)	Final concentration (mg/ml)
				C	protein A	3	0.5
T2	30G10	0.5	0.25
				T1	35E2	1.5	0.75

4.3 Process testing

Different parameter combinations are set, and the optimal process of the test strip is explored.

Watch 13

TABLE 14

Process A120 and Process A122 are preferred, with Process A122 being the final choice. When the mixing proportion of Italian honey in the Chinese honey is 20%, the Italian honey detection line (T1 line) still develops color, which indicates that the test strip has higher detection sensitivity. Sticking the sample pad, the coated nitrocellulose membrane and the water absorption pad onto a polyvinyl chloride (PVC) bottom plate at one time, cutting into strips with the width of 2-3mm, and packaging into a plastic shell to obtain the rapid detection test strip; drying and storing at 4 ℃ in dark.

4.4 detection

The test paper strip is used for detecting the Chinese honey, the Italian honey and the Chinese honey sample doped with the Italian honey in different proportions, correct results are obtained within 5-10min, and the specific results are shown in figure 4: the T1 line is not colored, and the C line and the T2 line are colored when the honey is pure. The C line, T1 line and T2 line all appear color when pure honey is obtained. When the honey is mixed into the Chinese honey, the C line, the T1 line and the T2 line are all colored, and the test paper strip can detect the mixing of 20% of the honey, so that the test paper strip is a reliable tool for quickly identifying the Chinese honey.

Example 5: application of rapid detection test strip

The application of the colloidal gold test strip for the authenticity of the Chinese honey and honey Italian bee species comprises the following steps:

1. taking out the test strip, and returning to room temperature;

2. preparation of honey sample detection solution: mixing a honey sample with pure water in a mass ratio of 1:10, diluting and uniformly mixing;

3. the test paper strip is horizontally placed, 2 drops of diluted honey sample are added into the sample pad, and the result is read after 5-10 minutes;

4. and (5) judging a result: the results are valid when the C line is displayed as a red line. When the T1 and T2 lines are developed, the result is Italian honey or Italian honey is mixed, namely the sample to be detected contains Italian MRJP2 protein; when the T1 line is not colored and the T2 line is colored, the result is Chinese bee honey, and the sample to be detected only contains the Chinese bee MRJP2 but not the Apis MRJP2; neither the T1 nor T2 lines developed, resulting in a syrup, i.e., the sample contained neither apis cerana MRJP2 nor apis cerana MRJP2. When the C line did not develop color, the results were not valid.

Although the present invention has been described in detail with reference to the above embodiments, the scope of the present invention is not limited thereto, and modifications may be made to the aspects of the present invention without departing from the center, which fall within the scope of the present invention.

Claims

1. The colloidal gold test strip for simultaneously detecting the sources of the apis mellifera and the apis mellifera species is characterized in that the test strip contains a specific apis mellifera MRJP2 monoclonal antibody and a specific apis cerana MRJP2 monoclonal antibody, the specific apis mellifera MRJP2 monoclonal antibody is obtained by secreting a 35E2 cell strain, and the preservation number of the 35E2 cell strain is as follows: CCTCC NO: C2022208, the specific Apis MRJP2 monoclonal antibody is obtained by the secretion of 30G10 cell strain, the preservation number of the 30G10 cell strain is as follows: CCTCC NO: C2022209.

2. The utility model provides a colloidal gold test paper strip of honey and Italian honey bee kind source in simultaneous detection which characterized in that, the test paper strip comprises absorption pad, nitrocellulose membrane, gold mark pad, sample pad, polyvinyl chloride bottom plate, and absorption pad, nitrocellulose membrane, gold mark pad, sample pad from the top down paste in proper order on the polyvinyl chloride bottom plate, are equipped with Italian honey detection line T1 on nitrocellulose membrane, well honey detection line T2 and matter accuse line C, and C line, T1 line and T2 line are respectively 5mm apart from each other, pack into plastic casing.

3. The method for preparing the colloidal gold test strip of claim 1 or 2, which is characterized by comprising the following steps:

(1) Antigen separation and purification: dialyzing and purifying royal jelly of apis cerana and apis mellifera by using a 14kDa dialysis bag to obtain a royal jelly major protein MRJPS crude extract; purifying antigen protein by column chromatography to obtain Apis cerana MRJP2 protein and Apis cerana MRJP2 protein which are used as antigens of subsequently immunized mice;

(2) Mouse immunization: using purified Chinese bee MRJP2 protein and Apis mellifera MRJP2 protein as antigens for immunization, selecting female BALB/c mice with 8 weeks old as immunized animals, and selecting mice with high titer to strengthen the immunization once 3 days before fusion;

(3) Fusion of mouse splenocytes with myeloma cells: selecting a cloning pore with a good cell state from fused cells 3B2, 31E2, 27D12, 1C1, 35C4 and 35E2 of Italian MRJP2 respectively to perform secondary subcloning according to specificity for Italian honey, and selecting a cloning pore with a good cell state from fused cells 13A5, 30G10, 25D10 and 11E7 of Italian MRJP2 respectively to perform secondary subcloning according to specificity for Italian honey;

(4) Preparation of rabbit anti-apis mellifera protein polyclonal antiserum: using the purified apis cerana MRJP2 and apis mellifera MRJP2 proteins as antigens for immunization to immunize New Zealand white rabbits, preparing polyclonal antibodies capable of simultaneously identifying apis cerana MRJP2 and apis mellifera MRJP2, and using the polyclonal antibodies as coating antibodies of the colloidal gold test strips;

(5) Pairing and marking of apis mellifera and apis mellifera honey monoclonal antibodies: the specific apis mellifera MRJP2 monoclonal antibody selects a 35E2 cell strain, and the deposit number of the 35E2 cell strain is as follows: the CCTCC NO is C2022208, monoclonal antibody secreted by 35E2 cell strain has strong specificity, and has NO cross reaction with the Chinese bee MRJP2, the specific Chinese bee MRJP2 monoclonal antibody selects 30G10 cell strain, the preservation number of the 30G10 cell strain is as follows: CCTCC NO: C2022209;

(6) Preparing the colloidal gold test strip:

(a) Preparing a colloidal gold solution: preparing a gold chlorate aqueous solution into a colloidal gold solution containing colloidal gold particles with the particle size of 20 nm;

(b) Colloidal gold-labeled antibody: the optimal pH value of the gold-labeled antibody is 8.5;

(c) Coating a C line, a T1 line and a T2 line on a nitrocellulose membrane, wherein the C line is a quality control line, a commercial goat anti-mouse lgG is coated, the T1 line is an Italian honey detection line, the T2 line is a Membristian honey detection line, the T1 line and the T2 line are coated with rabbit polyclonal antibodies capable of identifying Membristian MRJP2 and Italian MRJP2 simultaneously, a detection antibody of the T1 line is a specific Italian MRJP2 monoclonal antibody, a detection antibody of the T2 line is a Membristian MRJP2 monoclonal antibody, and the C line, the T1 line and the T2 line are respectively 5mm apart; drying at room temperature to obtain a coated nitrocellulose membrane;

(d) Assembling the colloidal gold immunochromatographic rapid detection test strip: and sequentially sticking the absorption pad, the nitrocellulose membrane, the gold label pad and the sample pad to a polyvinyl chloride bottom plate, cutting into strips with the width of 2-3mm, putting into a plastic shell to obtain the rapid detection test strip, and drying and storing at 4 ℃ in a dark place.

4. The use of the colloidal gold test strip of claim 1 in the detection of Chinese honey and honey bee species.

5. The use of claim 4, wherein the use comprises detection of authenticity of species sources of apis mellifera honey and apis mellifera honey, detection of apis mellifera honey adulteration, and detection of apis mellifera and apis mellifera honey related products.

6. The use according to claim 4, characterized by the fact that it is achieved by the following steps:

(1) Taking out the test strip, and returning to room temperature;

(3) The test paper strip is horizontally placed, 2 drops of diluted honey sample are added into the sample hole, and the result is read after 5-10 minutes;

(4) And (5) judging a result: when the C line is displayed as a red line, the result is effective, and when the T1 line and the T2 line are developed, the result is Italian honey or Italian honey is mixed, namely the sample to be detected contains Italian MRJP2; when the T1 line is not colored and the T2 line is colored, the result is Chinese bee honey, and the sample to be detected only contains the Chinese bee MRJP2 but not the Apis cerana MRJP2; neither the T1 nor T2 lines developed, resulting in a syrup, i.e., the sample contained neither apis cerana MRJP2 nor apis mellifera MRJP2; when the C line did not develop color, the results were not valid.