Small molecule detection kit

Abstract

The invention belongs to the field of rapid detection, and provides a small molecule detection kit. A traditional immunochromatography competitive inhibition mode for detecting small molecules is converted into a double-antibody sandwich mode through a conversion column and a universal mouse IgG immunochromatography test strip. A reverse reading signal on the detection line is converted into a positive reading signal, so that a signal can appear on the detection line of the test strip when trace small molecules exist, and compared with a traditional competitive immunochromatography method, the method has the characteristic of high detection sensitivity. The conversion column is formed by covalently coupling a target small molecule or an artificial antigen thereof with agarose or cellulose gel and then filling; the mouse IgG immunochromatography test strip consists of five parts, namely filter paper, a sample pad, a binding release pad (a probe with signal output capability is fixed, namely colloidal gold or a fluorescent microsphere nano material of a marked anti-mouse IgG monoclonal antibody 1), an NC (nitrocellulose) membrane (an anti-mouse IgG monoclonal antibody 2 is fixed on a detection line, and Protein G is fixed on a control line) and absorbent paper.

Description

Technical Field

[0001] The present invention relates to the field of rapid detection, and in particular to a positive read mode competitive immunochromatography small molecule rapid detection method based on a conversion column. Technical Background

[0002] Immunochromatography is a common method for rapid on-site screening because of its advantages of simple operation, rapidity and strong resistance to matrix interference. According to its detection principle, immunochromatography can be divided into two categories: double antibody sandwich method and competitive inhibition method. Double antibody sandwich method is usually used for macromolecules with multiple antigenic determinants, such as proteins. The signal strength on the detection line is proportional to the concentration of the analyte. When a very small amount of the analyte is present, a weak band that can be discerned by the naked eye can be formed; when detecting small molecules, because small molecules cannot bind to more than one antibody at the same time, competitive inhibition method is usually used. The signal strength on the detection line is inversely proportional to the concentration of the analyte. The higher the content of small molecules, the weaker the detection line signal. Qualitative analysis often uses the complete disappearance of the detection line signal as a positive judgment standard. This mode requires a large number of small molecules to completely block the detection line signal. Therefore, the detection sensitivity is not high, and it is difficult to meet the detection of small molecules when trace amounts are present. If the traditional small molecule detection mode can be converted to the "double antibody sandwich" mode for detecting macromolecules, the sensitivity of small molecule detection can be significantly improved.

[0003] Based on the above situation, we use the conversion column to convert the competitive inhibition mode of detecting small molecules into the double antibody sandwich mode of detecting large molecules, so that the small molecules with a molecular weight of hundreds are converted into immunoglobulin G (IgG) with a molecular weight of 150,000, which greatly improves the sensitivity of small molecule detection. At the same time, we designed a simple paper bracket to replace the traditional iron stand or plastic bracket to achieve vertical fixation of the conversion column, which not only saves costs but is also environmentally friendly. Summary of the invention

[0004] The present invention provides a small molecule detection kit, which mainly comprises four parts: mouse IgG monoclonal antibody of target small molecule, conversion column, universal IgG detection test strip and environmentally friendly paper support.

[0005] Mouse IgG monoclonal antibodies against target small molecules can specifically bind to the target small molecules.

[0006] The conversion column is filled with a target small molecule (or its artificial antigen) after covalent coupling with a gel with a supporting function such as agarose or cellulose, and includes a top sealing film, a bottom stopper and a receiving dish.

[0007] The universal test strip consists of five parts: filter paper, sample pad, binding release pad, NC membrane and absorbent paper. Among them, the binding release pad is fixed with a probe with signal output capability (such as colloidal gold or fluorescent microspheres labeled with anti-mouse IgG monoclonal antibody 1), the NC membrane detection line is fixed with anti-mouse IgG monoclonal antibody 2, and the control line is fixed with Protein G. The anti-mouse IgG antibody can be an anti-mouse IgG antibody of animals such as cattle, sheep, rabbits, and donkeys, and the anti-mouse IgG monoclonal antibodies 1 and 2 are a pair of paired antibodies for detecting IgG.

[0008] Environmentally friendly paper brackets are used to fix the conversion column vertically. There are two designs: one is a hard paper sheet that can be folded into a bracket through four dotted lines, called a four-legged foldable bracket ( Figure 1 ); the other is a hard paper with adhesive and round holes, called an adhesive stand ( Figure 2 ), fold the paper in half, remove the adhesive protective film, and stick it on any fixed vertical surface, such as the outer packaging box of the test kit, with the sticking position about 2 cm vertically above the desktop.

[0009] Specifically, the foldable bracket comprises a connecting body (11) and a plurality of supporting legs (12) evenly distributed on the connecting body (11); the connecting body (11) is provided with a first fixing hole (13), and the first fixing hole (13) is used to fix the conversion column; the plurality of supporting legs (12) are configured to generate a height difference between the supporting surfaces of the connecting body (11) and the bottom of the supporting legs (12) after being folded in the same direction.

[0010] Specifically, the adhesive bracket comprises an adhesive portion (22) and a fixing portion (23) located at both ends of the bending portion (21); the fixing portion (23) is provided with a second fixing hole (24), and the second fixing hole (24) is used to fix the conversion column; the adhesive portion (22) is provided with an adhesive piece (25) which can be adhered to a vertical surface and is used to fix the adhesive bracket; the bending portion (21) is used to bend the adhesive bracket so that the conversion column remains in a vertical state after passing through the second fixing hole (24).

[0011] The method of using the small molecule detection kit is as follows: open the kit, take out the environmentally friendly paper support, fold it and place it or stick it in place; take out the conversion column, insert it into the small hole of the paper support, place the receiving dish underneath, open the bottom stopper and the top sealing film; take out the test tube containing the target small molecule mouse IgG monoclonal antibody dry powder, mix it evenly with 200μL of the sample solution to be tested, and then drip it into the conversion column; after 2 minutes, insert one side of the sample pad of the test strip into the receiving dish, and after 10 minutes, take out the test strip for observation. If a band appears, it means that the test solution contains the target small molecule, and the content of the target small molecule can also be determined by the test strip signal reader.

[0012] The present invention includes the following technical solutions:

[0013] (1) Preparation of conversion column

[0014] Weigh 30g of CNBr-activated Sepharose gel powder, suspend it in 250mL of 1mM hydrochloric acid solution, and obtain 100mL of sol after complete swelling; wash it with 250mL of 1mM hydrochloric acid in a suction funnel, and then rinse it 5 times with 1L of a combination solution containing 0.1M sodium bicarbonate and 0.5M sodium chloride; then slowly stir the gel with an equal volume of 2mg / mL of small molecule complete antigen at room temperature for 3 hours, and then add 200mL of 50mg / mL bovine serum albumin (BSA) and slowly stir it at room temperature for 1.5 hours; transfer to a suction funnel, and fully wash it 5 times with 15 times the volume of 0.01M phosphate buffer solution (PBS), and the last time with 0.01M containing 0.3% sodium azide (NaN3) After washing with PBS and draining, the gel was loaded into a conversion column, the size and shape of which were similar to a 200 μL pipette tip. Two gaskets (upper gasket 321 and lower gasket 322) were placed on the upper and lower sides to fix the gel (33). The bottom was sealed with a stopper (34) and the top was sealed with a film (31). Figure 3 ).

[0015] (2) Preparation of universal test strips

[0016] The universal test strip consists of five parts: filter paper, sample pad, binding release pad (fixed with nanoprobes with signal output capability, such as colloidal gold or fluorescent microspheres labeled with anti-mouse IgG monoclonal antibody 1), NC membrane (detection line fixed with anti-mouse IgG monoclonal antibody 2, quality control line fixed with Protein G) and absorbent paper.

[0017] a. Preparation of sample pad

[0018] The glass fiber paper was soaked in a buffer solution containing 1.0% BSA, 0.25% Tween-20 and 20 mM sodium borate (0.1% (w / v) NaN3, pH 8.0) and dried naturally at room temperature.

[0019] b. Preparation of bonding release pad

[0020] The polyester fiber membrane was soaked with 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes and dried at 37°C overnight.

[0021] The steps of labeling anti-mouse IgG monoclonal antibody 1 with a nanoprobe having signal output capability (such as colloidal gold and fluorescent microspheres) are as follows:

[0022] (1) Colloidal gold labeled anti-mouse IgG monoclonal antibody 1

[0023] 1 mL of 1% HAuCl4 solution was added to 99 mL of pure water and heated to boiling. 2.3 mL of 1% sodium citrate solution was added under constant stirring, and the boiling was maintained for 10 minutes. After cooling, the pH was adjusted to 6.5-7.8.

[0024] 1 mL of 1 mg / mL anti-mouse IgG monoclonal antibody 1 was added to 200 mL of the above colloidal gold solution, and the mixture was slowly stirred at room temperature for 2 hours. 100 mL of 15% BSA aqueous solution was added to the mixture, and the mixture was slowly stirred at room temperature for 1.5 hours. After centrifugation at 4°C for 10 minutes (centrifugal force of 4446 g), the supernatant was discarded, and the precipitate was resuspended in 10 mL of phosphate buffer (PB) (containing 25% sucrose, 0.1% BSA and 0.1% isothiazolinone preservative (ProClin 300), pH 7.4), and stored at 4°C for future use.

[0025] (2) Fluorescent microsphere labeled anti-mouse IgG monoclonal antibody 1

[0026] Take 100 μL fluorescent microspheres (10 mg / mL) and vortex mix with 20 mL MES buffer (0.05 mM, pH 6.0); add cross-linking agents carbodiimide and N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared and used immediately) in sequence, and react at room temperature for 15 minutes; centrifuge at 4°C for 20 minutes (centrifugal force 7000g), remove the supernatant; add 20 mL boric acid buffer (10 mM, pH 8.0), vortex mix, and perform ultrasonic treatment if necessary; then add 150 μg antibody, mix, react at room temperature for 2 hours, and then add 5 mL of 10% BSA. Mix well and react at room temperature for 30 minutes; centrifuge at 4°C for 30 minutes (centrifugal force of 7000g) and remove the supernatant, wash twice with 10mM boric acid buffer (pH 8.0); add 3mL preservation solution (2% sucrose, 1% alginate, 1% polyethylene glycol 20000, 1% casein and 0.4% Tween-20) and store at 4°C.

[0027] The IgG detection probe prepared above was sprayed on the binding release pad at 10 μL / cm, dried in a vacuum drying oven at 37° C. for 6 hours, and stored in a dry condition at room temperature.

[0028] c. Preparation of test line and quality control line on NC membrane

[0029] 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G were sprayed at 0.75 μL / cm2 on the detection line and quality control line, respectively, with a distance of 5 mm between the two lines. The NC membrane was placed in a 37°C constant temperature drying oven for 12 hours and stored in a dry condition at room temperature.

[0030] d. Assembly of test strips

[0031] The NC membrane was adhered to the center of the plastic backing plate, and the filter paper, sample pad and binding release pad were adhered to one end of the NC membrane in sequence, and the other end was adhered to absorbent paper. Subsequently, it was cut into single strips with a width of 4 mm, sealed in a light-shielding bag, and stored in a dry condition at room temperature.

[0032] (3) Use of the kit

[0033] The sample extract was appropriately diluted and the pH was adjusted to the range of 6.5 to 8.0.

[0034] Take 200 μL of the above test solution and add it into the test tube containing the target small molecule IgG monoclonal antibody dry powder, and mix for a while.

[0035] Take out the conversion column and insert it into the environmentally friendly paper holder, place a receiving dish underneath, tear off the sealing film on the conversion column, remove the bottom plug, pour in the mixed liquid, wait for about 2 minutes (when the liquid no longer drips continuously), insert the test strip into the receiving dish, and observe the detection line band after 10 minutes. If a band appears, it means that the target small molecule is contained in the test object, and the content of the target small molecule can also be determined by the test strip signal reader.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The small molecule detection kit provided by the present invention converts the traditional immunochromatographic competitive inhibition mode of small molecule detection into a double antibody sandwich mode through a conversion column and a universal immunochromatographic test strip. The reverse reading signal on the detection line is converted into a forward reading signal, so that when a small molecule is present in trace amounts, a signal can appear on the detection line of the test strip. Compared with the traditional competitive immunochromatographic method, it has the characteristics of high detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the structure of the foldable bracket provided by the present invention;

[0039] Figure 2 A schematic diagram of the structure of the adhesive bracket provided by the present invention;

[0040] Figure 3 A schematic diagram of a conversion column provided by the present invention;

[0041] Figure 4 A schematic diagram of the qualitative determination test strip results provided in Example 1 of the present invention;

[0042] Figure 5 A schematic diagram of the qualitative determination test strip results provided in Example 2 of the present invention;

[0043] Figure 6 This is a schematic diagram of the qualitative determination test strip results provided in Example 3 of the present invention.

[0044] Icons: 11-connecting body; 12-supporting foot; 13-first fixing hole; 21-bending part; 22-adhesive part; 23-fixing part; 24-second fixing hole; 25-adhesive piece; 31-film; 321-upper gasket; 322-lower gasket; 33-gel; 34-plug. DETAILED DESCRIPTION

[0045] In order to make the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] The detection antigens and antibodies involved in the examples were prepared by our research group; reagents such as chloroauric acid, BSA, and cyanogen bromide-activated agarose gel 4B were from Sigma; quantum dot fluorescent microspheres and test strip related raw materials including NC membrane (model CN95), sample pad, binding release pad, filter paper, absorbent paper, sticky card board and plastic card shell were from Jiangxi Weibang Biotechnology Co., Ltd.

[0047] Example 1: Preparation and use of a detection kit for enrofloxacin (ENR) in eggs

[0048] 1. Colloidal gold labeled anti-mouse IgG monoclonal antibody 1 (colloidal gold detection probe)

[0049] (1) Preparation of colloidal gold

[0050] 1 mL of 1% HAuCl4 solution was added to 99 mL of pure water and heated to boiling. 2.3 mL of 1% sodium citrate solution was added under constant stirring, and the boiling was maintained for 10 minutes. After cooling, the pH was adjusted to 6.5-7.8.

[0051] (2) Antibody labeling

[0052] 1 mL of 1 mg / mL anti-mouse IgG monoclonal antibody 1 was added to 200 mL of the above colloidal gold solution, and the mixture was slowly stirred at room temperature for 2 hours. 100 mL of 15% BSA aqueous solution was added to the mixture, and the mixture was slowly stirred at room temperature for 1.5 hours. After centrifugation at 4°C for 10 minutes (centrifugal force of 4446 g), the supernatant was discarded, and the precipitate was resuspended in 10 mL of phosphate buffer (PB) (containing 25% sucrose, 0.1% BSA and 0.1% isothiazolinone preservative (ProClin 300), pH 7.4), and stored at 4°C for future use.

[0053] 2. Preparation of Enrofloxacin Artificial Antigen (ENR-BSA):

[0054] The coupling method is the carbodiimide method, the coupling protein is bovine serum albumin (BSA), the coupling ratio is 1:60, and after coupling, it is dialyzed and purified to obtain ENR-BSA.

[0055] 3. Preparation of ENR Conversion Column

[0056] Weigh 30g of CNBr-activated Sepharose gel powder, suspend it in 250mL of 1mM hydrochloric acid solution, and obtain 100mL of sol after complete swelling; wash it with 250mL of 1mM hydrochloric acid in a suction funnel, and then rinse it 5 times with a binding solution (1L) containing 0.1M sodium bicarbonate and 0.5M sodium chloride; then slowly stir the gel with an equal volume of 2mg / mL ENR-BSA (based on BSA amount) at room temperature for 3 hours, then add an equal volume of 50mg / mL bovine serum albumin (BSA) and slowly stir it at room temperature for 1.5 hours; transfer to a suction funnel, wash it thoroughly with 15 times the volume of 0.01M phosphate buffer solution (PBS) for 5 times, and wash it with 0.01M PBS containing 0.3% sodium azide (NaN3) for the last time, and then load it into a conversion column after drying. The size and shape of the conversion column are similar to those of a 200μL pipette tip, the bottom end is sealed with a stopper, and the top end is sealed with a film.

[0057] 4. Preparation of universal test strips

[0058] (1) Preparation of sample pad

[0059] The glass fiber paper was soaked in a buffer solution containing 1.0% BSA, 0.25% Tween-20 and 20 mM sodium borate (0.1% (w / v) NaN3, pH 8.0) and dried naturally at room temperature.

[0060] (2) Preparation of bonding release pad

[0061] The polyester fiber membrane was soaked with 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes and dried at 37°C overnight.

[0062] The prepared colloidal gold probe solution was sprayed on the binding release pad at 10 μL / cm, dried in a vacuum drying oven at 37° C. for 6 hours, and stored in a dry condition at room temperature.

[0063] (3) Preparation of test line and quality control line on NC membrane

[0064] 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G were sprayed at 0.75 μL / cm2 on the detection line and quality control line, respectively, with a distance of 5 mm between the two lines. The NC membrane was placed in a 37°C constant temperature drying oven for 12 hours and stored in a dry condition at room temperature.

[0065] 5. Assembly of test strips

[0066] The NC membrane was adhered to the center of the plastic backing plate, and the filter paper, sample pad, and binding release pad were adhered to one end of the NC membrane in sequence, and the other end was adhered to absorbent paper. Subsequently, it was cut into single strips with a width of 4 mm, sealed in a light-shielding bag, and stored in a dry condition at room temperature.

[0067] 6. Detection of ENR in eggs

[0068] (1) The pretreatment was carried out in accordance with the Announcement No. 781 of the Ministry of Agriculture and Rural Affairs-6-2006 Determination of Fluoroquinolone Residues in Eggs by High Performance Liquid Chromatography.

[0069] (2) Take 200 μL of the above sample treatment solution and add it to a test tube containing 20 μg of ENR monoclonal antibody dry powder and mix well.

[0070] (3) Take out the conversion column and insert it into the environmentally friendly paper holder, tear off the sealing film on the conversion column, pull out the small plug at the bottom, place a receiving dish underneath, pour in the mixed liquid, and wait for about 1 minute (when the liquid no longer drips continuously), insert one end of the test strip sample pad into the receiving dish. After 10 minutes, the qualitative and quantitative determination of ENR in eggs can be achieved.

[0071] Qualitative judgment

[0072] a. First check whether the C line is colored. If not, it means the test strip is invalid.

[0073] b. If the C line is normal, press Figure 4 Determine the result.

[0074] Note: GB 31650 Announcement No. 594 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China issued the "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food" (GB 31650.1-2022), which stipulates that the maximum residue limit of ENR in eggs is 10μg / kg.

[0075] Positive judgment value: When the concentration of ENR is not less than 10μg / kg, the kit can detect positive results.

[0076] Quantitative determination

[0077] a. Turn on the colloidal gold signal analyzer, insert the test card into the test card slot, click "Test" on the instrument interface, and the instrument will automatically give the quantitative test results;

[0078] b. If the test result exceeds the upper limit of the quantitative detection limit, it can be diluted with diluent and tested again.

[0079] Example 2: Preparation and use of a detection kit for sulfadimethoxine (SMZ) in eggs

[0080] 1. CdSe / ZnS red quantum dot fluorescent microspheres labeled mouse IgG monoclonal antibody 1 (quantum dot fluorescent microsphere detection probe)

[0081] Take 10 μL quantum dot fluorescent microspheres (10 mg / mL) and surface carboxyl-modified CdSe / ZnS quantum dot fluorescent microspheres (excitation 365 nm, emission 630 nm), and vortex mix with 2 mL MES buffer (0.05 mM, pH 6.0); add crosslinking agents carbodiimide and N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared and used immediately) in sequence, and react at room temperature for 15 minutes; centrifuge at 4°C for 20 minutes (centrifugal force 7000g), remove the supernatant; add 2 mL boric acid buffer (10 mM, pH 8.0), vortex mix, and perform ultrasonic treatment if necessary; then add 15 μg anti-mouse IgG monoclonal antibody 1 and mix, and add 500 μL of 10% BSA after reacting at room temperature for 2 hours. Mix well and react at room temperature for 30 minutes; centrifuge at 4°C for 30 minutes (centrifugal force of 7000g) and remove the supernatant, wash twice with 10mM boric acid buffer (pH 8.0); add 300μL preservation solution (2% sucrose, 1% alginate, 1% polyethylene glycol 20000, 1% casein and 0.4% Tween-20) and store at 4°C.

[0082] 2. Preparation of sulfamethoxazole artificial antigen (SMZ-BSA):

[0083] The coupling method is diazotization method, the coupling protein is bovine serum albumin (BSA), the coupling ratio is 1:60, and after coupling, dialysis purification is performed to obtain SMZ-BSA.

[0084] 3. Preparation of SMZ Conversion Column

[0085] Weigh 30g CNBr activated Sepharose gel powder, suspend in 250mL of 1mM hydrochloric acid solution, and obtain 100mL sol after complete swelling; wash with 250mL volume of 1mM hydrochloric acid in a suction funnel, and then rinse 5 times with a combination solution (how much) containing 0.1M sodium bicarbonate and 0.5M sodium chloride; then slowly stir the gel with an equal volume of 2mg / mL SMZ-BSA at room temperature for 3 hours, then add 50mg / mL bovine serum albumin (BSA) (how much) and slowly stir at room temperature for 1.5 hours; transfer to a suction funnel, fully wash with 15 times volume of 0.01M phosphate buffer solution (PBS) for 5 times, and wash with 0.01M PBS containing 0.3% sodium azide (NaN3) for the last time, and load into a conversion column after drying. The size and shape of the conversion column are similar to a 200μL pipette tip, the bottom end is sealed with a stopper, and the top end is sealed with a film.

[0086] 4. Preparation of universal test strips

[0087] (1) Preparation of sample pad

[0088] The glass fiber paper was soaked in a buffer solution containing 1.0% BSA, 0.25% Tween-20 and 20 mM sodium borate (0.1% (w / v) NaN3, pH 8.0) and dried naturally at room temperature.

[0089] (2) Preparation of bonding release pad

[0090] The polyester fiber membrane was soaked with 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes and dried at 37°C overnight.

[0091] The prepared quantum dot fluorescent microsphere detection probe solution was sprayed on the binding release pad at 10 μL / cm, placed in a vacuum drying oven at 37° C. for 6 hours, and stored in a dry condition at room temperature.

[0092] (3) Preparation of test line and quality control line on NC membrane

[0093] 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G were sprayed at 0.75 μL / cm2 on the detection line and quality control line, respectively, with a distance of 5 mm between the two lines. The NC membrane was placed in a 37°C constant temperature drying oven for 12 hours and stored in a dry condition at room temperature.

[0094] 5. Assembly of test strips

[0095] The NC membrane was adhered to the center of the plastic backing plate, and the sample pad, binding release pad, and filter paper were adhered to one end of the NC membrane in sequence, and the other end was adhered to absorbent paper. Subsequently, it was cut into single strips with a width of 4 mm, sealed in a light-shielding bag, and stored in a dry condition at room temperature.

[0096] 6. Detection of SMZ in Eggs

[0097] (1) After the egg samples are shelled, they are homogenized in a homogenizer at 500 r / min for 20 s to fully mix the egg white and egg yolk; 1.0 g of the homogenate is weighed and placed in a centrifuge tube, 5 mL of phosphate buffer is added, and the tube is gently shaken manually for 20 minutes to avoid emulsification; the tube is centrifuged at 4000 r / min for 10 minutes at room temperature, and the supernatant is diluted 4 times with phosphate buffer for testing.

[0098] (2) Take 200 μL of the above sample solution and add it to a test tube containing 20 μg of SMZ monoclonal antibody dry powder and mix well.

[0099] (3) Take out the conversion column and insert it into the environmentally friendly paper holder, tear off the sealing film on the conversion column, pull out the small plug at the bottom, place a receiving dish underneath, pour in the mixed liquid, and wait for about 1 minute (when the liquid no longer drips continuously), insert one end of the test strip sample pad into the receiving dish. After 10 minutes, the qualitative and quantitative detection of SMZ in eggs can be achieved.

[0100] Qualitative judgment

[0101] Use a 365nm LED lamp to illuminate the NC film of the test strip.

[0102] a. First check whether the C line is colored. If not, it means the test strip is invalid.

[0103] b. If the C line is normal, press Figure 5 Determine the result.

[0104] Note: GB 31650 Announcement No. 594 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China | "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food" (GB 31650.1-2022) stipulates that the maximum residue limit of SMZ in eggs is 10μg / kg.

[0105] Positive judgment value: When the concentration of SMZ is not less than 10μg / kg, the kit can detect positive results

[0106] Quantitative determination

[0107] a. Open the test strip fluorescence signal analyzer, insert the test card into the test card slot, click "Test" on the instrument interface, and the instrument will automatically give the quantitative test results;

[0108] b. If the test result exceeds the upper limit of the quantitative detection limit, it can be diluted with diluent and tested again.

[0109] Example 3: Preparation and use of a detection kit for progesterone (PROG) in whole blood, serum and plasma samples

[0110] 1. Green aggregation-induced emission (AIE) fluorescent microspheres labeled with anti-mouse IgG monoclonal antibody 1 (AIE fluorescent microsphere detection probe)

[0111] Take 10 μL of green AIE fluorescent microspheres (10 mg / mL; excitation 365 nm, emission 510 nm), vortex mix with 2 mL of MES buffer (0.05 mM, pH 6.0); add cross-linking agents carbodiimide and N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared and used immediately) in sequence, react at room temperature for 15 minutes; centrifuge at 4°C for 20 minutes (centrifugal force 7000g), remove the supernatant; add 2 mL of boric acid buffer (10 mM, pH 8.0), vortex mix, and perform ultrasonic treatment if necessary; then add 15 μg of anti-mouse IgG monoclonal antibody 1 and mix, react at room temperature for 2 hours, and then add 500 μL of 10% BSA. Mix well and react at room temperature for 30 minutes; centrifuge at 4°C for 30 minutes (centrifugal force of 7000g) and remove the supernatant, wash twice with 10mM boric acid buffer (pH 8.0); add 300μL preservation solution (2% sucrose, 1% alginate, 1% polyethylene glycol 20000, 1% casein and 0.4% Tween-20) and store at 4°C.

[0112] 2. Preparation of sulfadimethoxine artificial antigen (PROG-BSA):

[0113] The coupling method was the carbodiimide method, the coupling protein was bovine serum albumin (BSA), the coupling ratio was 1:60, and after coupling, it was dialyzed and purified to obtain PROG-BSA.

[0114] 3. Preparation of PROG Conversion Column

[0115] Weigh 30g of CNBr-activated Sepharose gel powder, suspend it in 250mL of 1mM hydrochloric acid solution, and obtain 100mL of sol after complete swelling; wash it with 250mL of 1mM hydrochloric acid in a suction funnel, and then rinse it 5 times with 1L of a combination solution containing 0.1M sodium bicarbonate and 0.5M sodium chloride; then slowly stir the gel with an equal volume of 2mg / mL PROG-BSA at room temperature for 3 hours, then add an equal volume of 50mg / mL bovine serum albumin (BSA) and slowly stir it at room temperature for 1.5 hours; transfer to a suction funnel, and wash it thoroughly with 15 times the volume of 0.01M phosphate buffer solution (PBS) for 5 times, and wash it with 0.01M PBS containing 0.3% sodium azide (NaN3) for the last time, and then load it into a conversion column after drying. The size and shape of the conversion column are similar to a 200μL pipette tip, the bottom end is sealed with a stopper, and the top end is sealed with a film.

[0116] 4. Preparation of universal test strips

[0117] (1) Preparation of sample pad

[0118] The glass fiber paper was soaked in a buffer solution containing 1.0% BSA, 0.25% Tween-20 and 20 mM sodium borate (0.1% (w / v) NaN3, pH 8.0) and dried naturally at room temperature.

[0119] (2) Preparation of bonding release pad

[0120] The polyester fiber membrane was soaked with 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes and dried at 37°C overnight.

[0121] The prepared AIE fluorescent microsphere detection probe solution was sprayed on the binding release pad at 10 μL / cm, placed in a vacuum drying oven at 37°C for 6 hours, and stored in a dry condition at room temperature.

[0122] (3) Preparation of test line and quality control line on NC membrane

[0123] 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G were sprayed at 0.75 μL / cm2 on the detection line and quality control line, respectively, with a distance of 5 mm between the two lines. The NC membrane was placed in a 37°C constant temperature drying oven for 12 hours and stored in a dry condition at room temperature.

[0124] 5. Assembly of test strips

[0125] The NC membrane was adhered to the center of the plastic backing plate, and the sample pad, binding release pad, and filter paper were adhered to one end of the NC membrane in sequence, and the other end was adhered to absorbent paper. Subsequently, it was cut into single strips with a width of 4 mm, sealed in a light-shielding bag, and stored in a dry condition at room temperature.

[0126] 6. Detection of PROG in whole blood, serum and plasma samples

[0127] (1) Take the sample to be tested and add it to the sample diluent (the main component is Tris-HCl), mix well, and adjust the pH to the range of 6.5 to 8.0.

[0128] (2) Take 200 μL of the above sample solution and add it to a test tube containing 20 μg of PROG monoclonal antibody dry powder and mix well.

[0129] (3) Take out the conversion column and insert it into the environmentally friendly paper holder, tear off the sealing film on the conversion column, pull out the small plug at the bottom, place a receiving dish underneath, pour in the mixed liquid, wait for about 1 minute (when the liquid no longer drips continuously), insert the test strip into the receiving dish, and after 10 minutes, achieve qualitative and quantitative detection of PROG in whole blood, serum and plasma samples.

[0130] Qualitative judgment

[0131] Use a 365nm LED lamp to illuminate the NC film of the test strip.

[0132] a. First check whether the C line is colored. If not, it means the test strip is invalid.

[0133] b. If the C line is normal, press Figure 6 Determine the result.

[0134] Note: The normal values ​​of progesterone in different periods are: 0.6-1.9nmol / L in the follicular phase, 2.4-9.4nmol / L in the ovulatory phase, and 20.7-102.4nmol / L after ovulation.

[0135] Positive judgment value: When the concentration of PROG is not less than 0.6nmol / L, the kit can detect positive results.

[0136] Quantitative determination

[0137] a. Open the test strip fluorescence signal analyzer, insert the test card into the test card slot on the lower right, click "Test" on the instrument interface, and the instrument will automatically give the quantitative test results;

[0138] b. If the test result exceeds the upper limit of the quantitative detection limit, it can be diluted with diluent and tested again.

[0139] In summary, the present invention provides a kit for detecting small molecules based on the double antibody sandwich mode, which can realize multiple output modes such as colorimetry and fluorescence, and can be applied to sensitive detection of various small molecule targets.

Claims

1. A small molecule detection kit, mainly including mouse IgG monoclonal antibody of target small molecule, conversion column, universal test strip and environmentally friendly paper support.

2. According to the small molecule detection kit of claim 1, the small molecule analytes cover all small molecule substances that need to be detected in the fields of medical inspection, environmental protection and food safety testing.

3. A small molecule detection kit according to claim 1, wherein the conversion column is formed by covalently coupling the target small molecule or its artificial antigen with a gel having a supporting function, and is filled with a sealing film on the top, a stopper on the bottom, and an optional receiving dish; The gel having a supporting function includes agarose or cellulose.

4. A small molecule detection kit according to claim 1, wherein the universal test strip comprises filter paper, a sample pad, a binding release pad, an NC membrane and absorbent paper; in, A probe with signal output capability is fixed on the binding release pad, i.e., a nanomaterial labeled with anti-mouse IgG monoclonal antibody 1; anti-mouse IgG monoclonal antibody 2 is fixed on the NC membrane detection line, and Protein G is fixed on the control line; The nanomaterial with signal output capability includes colloidal gold or fluorescent microspheres; Anti-mouse IgG monoclonal antibody 1 and anti-mouse IgG monoclonal antibody 2 are a pair of antibodies for detecting IgG.

5. According to a small molecule detection kit according to claim 1, the environmentally friendly paper bracket includes the following two types: a foldable bracket or an adhesive bracket, which is used to vertically fix the conversion column when the kit is used.

6. A method for using a small molecule detection kit according to any one of claims 1 to 5, comprising: Open the test kit, take out the environmentally friendly paper support, and fold or stick the paper support to fix it; Take out the conversion column, insert it into the small hole of the paper holder, place the receiving dish underneath, open the bottom stopper and the top sealing film; add 200 μL of the sample solution to be tested into the test tube containing the target small molecule mouse IgG monoclonal antibody dry powder, mix evenly and drip into the conversion column; after 2 minutes, insert one side of the sample pad of the test strip into the receiving dish, and after 10 minutes, read the signal data of the detection line and the quality control line on the test strip through the test strip reader, and use the built-in standard curve to calculate the concentration of the target small molecule in the sample solution to be tested to achieve quantitative detection; qualitative analysis can be achieved by directly observing whether the test strip has a detection line.

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