A small molecule detection kit

Abstract

The present application belongs to the field of rapid detection, and provides a small molecule detection kit. The traditional detection of small molecules by immunochromatographic competition inhibition mode is converted into a double-antibody sandwich mode through a conversion column and a general mouse IgG immunochromatographic test strip. The reverse reading signal on the detection line is converted into a positive reading signal, so that the trace existence of small molecules can make the detection line of the test strip appear a signal, and compared with the traditional competitive immunochromatographic method, the present application has the characteristics of high detection sensitivity. The conversion column is filled and formed after the target small molecule or its artificial antigen is covalently coupled with agarose or cellulose gel; the mouse IgG immunochromatographic test strip is composed of five parts, i.e. filter paper, sample pad, combination and release pad (fixed with a probe having signal output capability, i.e. labeled anti-mouse IgG monoclonal antibody 1 colloidal gold or fluorescent microspherical nanomaterial), NC membrane (anti-mouse IgG monoclonal antibody 2 is fixed on the detection line, and Protein G is fixed on the control line), and water absorption paper.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rapid detection, in particular to a positive reading mode competitive immunochromatographic small molecule rapid detection method based on a conversion column. TECHNICAL BACKGROUND

[0002] The immunochromatographic method is a commonly used method for on-site rapid screening due to its advantages of simple operation, rapidness and strong anti-matrix interference capability. According to its detection principle, the immunochromatographic method can be divided into two categories: double-antibody sandwich method and competitive inhibition method. The double-antibody sandwich method is usually used for macromolecular substances with multiple antigenic determinants, such as proteins, and the signal strength on the detection line is proportional to the concentration of the test substance. When a trace amount of test substance exists, a weak band that can be distinguished by the naked eye can be formed. When detecting small molecules, the competitive inhibition method is usually used because small molecules cannot be bound to more than one antibody at the same time. The signal strength on the detection line is inversely proportional to the concentration of the test substance. The higher the content of the small molecule, the weaker the signal on the detection line. Qualitative analysis usually takes the complete disappearance of the detection line signal as the positive reading standard. This mode requires a large amount of small molecules to completely block the detection line signal, so the detection sensitivity is not high, and it is difficult to meet the detection of trace amounts of small molecules. 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 convert the competitive inhibition mode for detecting small molecules into the double-antibody sandwich mode for detecting macromolecules by using a conversion column, so that the detection of small molecules with a molecular weight of several hundred is converted to the detection of immunoglobulin G (IgG) with a molecular weight of 150,000, greatly improving the sensitivity of small molecule detection. At the same time, we designed a simple paper support to replace the traditional iron stand or plastic support to realize the vertical fixation of the conversion column, which not only saves cost, but also is environmentally friendly. SUMMARY

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

[0005] The mouse IgG monoclonal antibody of the target small molecule can specifically bind to the target small molecule.

[0006] The conversion column is filled and installed after the covalent coupling of the target small molecule (or its artificial antigen) and agarose or cellulose and other gels with supporting function, and contains a top sealing film, a bottom plug and a receiving dish.

[0007] The general test strip includes five parts of filter paper, sample pad, combination release pad, NC membrane and water absorption paper.

[0008] The environmental protection paper support is used for vertical fixation of the conversion column, and includes two designs: one is a hard paper sheet which can be folded into a support through four dashed lines, referred to as a four-legged foldable support Figure 1 ); and the other is a hard paper sheet with adhesive and a round hole, referred to as a pasting support Figure 2 ) which is folded in half and the adhesive protective film is removed, and is pasted on any fixable vertical surface, such as the outer packaging box of the kit, and the pasting position is about 2 cm from the vertical height of the desktop.

[0009] Specifically, the foldable support includes a connecting body (11) and a plurality of legs (12) uniformly distributed on the connecting body (11); the connecting body (11) is provided with a first fixing hole (13) for fixing the conversion column; and the plurality of legs (12) are configured to generate a height difference between the connecting body (11) and the support surface at the bottom of the leg (12) after folding in the same direction.

[0010] Specifically, the pasting support includes a pasting part (22) and a fixing part (23) at both ends of a bending part (21); the fixing part (23) is provided with a second fixing hole (24) for fixing the conversion column; the pasting part (22) is provided with a pasting member (25) which can be pasted on a vertical surface for fixing the pasting support; and the bending part (21) is used for bending the pasting support so that the conversion column remains in a vertical state after passing through the second fixing hole (24).

[0011] The use method of the small molecule detection kit is as follows: opening the kit, taking out the environmental protection paper support to fold or paste; taking out the conversion column, inserting it into the small hole of the paper support, placing the receiving dish below, opening the bottom plug and the top sealing film; taking out the test tube containing the target small molecule mouse IgG monoclonal antibody dry powder, mixing it with 200 muL of the sample solution, and then dropping it into the conversion column; after 2 minutes, inserting the sample pad of the test strip into the receiving dish, and after 10 minutes, taking out the test strip to observe, if there is a band, it indicates that the sample solution contains the target small molecule, and the content of the target small molecule can also be determined by the test strip signal reading instrument.

[0012] The present application comprises the following technical solutions:

[0013] (1) Preparation of the conversion column

[0014] Weigh 30 g of CNBr activated Sepharose gel dry powder, suspend in 250 mL of 1 mM hydrochloric acid solution, get 100 mL sol after complete swelling; in the suction filter funnel with 250 mL volume of 1 mM hydrochloric acid washing, and then use 1 L containing 0.1 M sodium bicarbonate and 0.5 M sodium chloride binding liquid rinse 5 times; then the gel with equal volume of 2 mg / mL small molecule whole antigen room temperature slow stirring 3 hours, then add 50 mg / mL bovine serum albumin (BSA) 200 mL room temperature slow stirring 1.5 hours; to the suction filter funnel, with 15 times volume of 0.01 M phosphate buffer solution (PBS) in 5 times fully washed, the last time with 0.01 M PBS containing 0.3% sodium azide (NaN3) washing, suction dry and put into the conversion column, the conversion column size and shape similar to 200 μL pipette tip, the upper and lower placed 2 gasket (upper gasket 321 and lower gasket 322) will be fixed gel (33), the bottom with plug (34) sealed, the top with film (31) sealed Figure 3 ).

[0015] (2) Preparation of the general test strip

[0016] The general test strip consists of filter paper, sample pad, conjugate pad (immobilize nano probe with signal output ability, such as colloidal gold or fluorescent microspheres labeled anti-mouse IgG monoclonal antibody 1), NC membrane (detection line immobilized anti-mouse IgG monoclonal antibody 2, and quality control line immobilized Protein G) and absorbent paper.

[0017] a. Preparation of the sample pad

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

[0019] b. Preparation of the conjugate pad

[0020] Soak the polyester fiber membrane in 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes, and dry overnight at 37°C.

[0021] The steps of labeling anti-mouse IgG monoclonal antibody 1 with nano probe with signal output ability (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 boiling was maintained for 10 minutes, and after cooling, the pH was adjusted to 6.5-7.8.

[0024] 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 boiling was maintained for 10 minutes, and after cooling, the pH was adjusted to 6.5-7.8.

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

[0026] Take 100 μL of fluorescent microspheres (10 mg / mL) and mix with 20 mL of MES buffer (0.05 mM, pH 6.0) by vortexing; then add the cross-linking agent carbodiimide and N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared fresh) in sequence, and react at room temperature for 15 minutes; centrifuge at 4°C for 20 minutes (centrifugal force 7000 g), and remove the supernatant; add 20 mL of boric acid buffer (10 mM, pH 8.0) and mix by vortexing, and if necessary, perform ultrasonic treatment; then add 150 μg of antibody, mix well, and react at room temperature for 2 hours, and then add 5 mL of 10% BSA. Mix well, react at room temperature for 30 minutes; centrifuge at 4°C for 30 minutes (centrifugal force 7000 g), and remove the supernatant, and wash twice with 10 mM boric acid buffer (pH 8.0); add 3 mL of storage solution (2% sucrose, 1% alginic acid, 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 at 10 μL / cm on the conjugate release pad, and dried in a vacuum drying oven at 37°C for 6 hours, and stored under room temperature drying conditions.

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

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

[0030] d. Assembly of test strip

[0031] The NC membrane is adhered to the center of the plastic backboard, and the filter paper, sample pad and combined release pad are sequentially adhered to one end of the NC membrane, and the water absorption paper is adhered to the other end. Then, a single strip with a width of 4 mm is cut, sealed in an opaque bag, and stored in a dry room.

[0032] (3) Use of the kit

[0033] The sample extraction solution is appropriately diluted, and the pH is adjusted to the range of 6.5-8.0.

[0034] 200 μL of the above-mentioned test solution is added to a test tube containing the target small molecule IgG monoclonal antibody dry powder, and mixed for a few moments.

[0035] The conversion column is taken out and inserted into the environmentally friendly paper support, the bottom is placed on the receiving dish, the sealing film on the conversion column is torn open, the bottom plug is pulled out, the mixed solution is poured in, about 2 minutes (when the liquid no longer continuously drops), the test strip is inserted into the receiving dish, and after 10 minutes, the detection line band is observed. If a band appears, it indicates that the target small molecule is contained in the test substance, and the content of the target small molecule can also be determined by the test strip signal reading instrument.

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

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

[0038] Figure 1 The structure schematic diagram of the foldable support provided by the present application;

[0039] Figure 2 The structure schematic diagram of the pasting support provided by the present application;

[0040] Figure 3 The schematic diagram of the conversion column provided by the present application;

[0041] Figure 4 The qualitative determination test strip result schematic diagram provided by Example 1 of the present application;

[0042] Figure 5 The qualitative determination test strip result schematic diagram provided by Example 2 of the present application;

[0043] Figure 6 The qualitative determination test strip result schematic diagram provided by Example 3 of the present application.

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

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

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

[0047] Example 1: Preparation and use of the 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, slowly stirred at room temperature for 2 hours, 100 mL of 15% BSA aqueous solution was added to the mixture, slowly stirred at room temperature for 1.5 hours, centrifuged at 4°C for 10 minutes (centrifugal force was 4446g), 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 standby.

[0053] 2. Preparation of enrofloxacin artificial antigen (ENR-BSA):

[0054] The coupling method is carbodiimide method, the coupling protein is bovine serum albumin (BSA), the coupling ratio is 1:60, and ENR-BSA is obtained after coupling and dialysis purification.

[0055] 3. Preparation of ENR conversion column

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

[0057] 4. Preparation of general test strip

[0058] (1) Preparation of sample pad

[0059] Soak the glass fiber paper 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 naturally air dry at room temperature.

[0060] (2) Preparation of conjugate release pad

[0061] Soak the polyester fiber membrane in 10 mM phosphate buffered saline containing 0.1% Tween-20 and 1% BSA (pH 7.4) for 5 minutes, and dry overnight at 37°C.

[0062] Spray the prepared colloidal gold probe solution on the conjugate release pad at a rate of 10 μL / cm, dry in a vacuum drying oven at 37°C for 6 hours, and store in room temperature dry conditions.

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

[0064] Spray 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G at a rate of 0.75 μL / cm on the test line position and the quality control line position respectively, and the distance between the two lines is 5 mm, place the NC membrane in a constant temperature drying oven at 37°C for 12 hours, and store in room temperature dry conditions.

[0065] 5. Assembly of test strip

[0066] The NC membrane is adhered to the center of the plastic backboard, and the filter paper, sample pad, and binding release pad are sequentially adhered to one end of the NC membrane, and the other end is adhered to the water absorption paper. Then, cut into single strips with a width of 4 mm, seal in light-shielding bags, and store in room temperature dry conditions.

[0067] 6. Detection of ENR in chicken eggs

[0068] (1) The pretreatment refers to the "Ministry of Agriculture and Rural Affairs No. 781 Announcement-6-2006 Determination of Fluoroquinolone Residues in Chicken 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 environmental paper support, tear off the sealing film on the conversion column, pull out the small plug below, place the receiving dish below, pour in the mixed solution, about 1 minute (when the liquid no longer continuously drips), insert the sample pad of the test strip into the receiving dish, and after 10 minutes, the qualitative and quantitative determination of ENR in chicken eggs is realized.

[0071] Qualitative determination

[0072] a. First, check if the C line is colored. If not, the test strip is invalid;

[0073] b. If the C line is normally colored, the result is determined according to Figure 4

[0074] Note: The maximum residue limit of ENR in chicken eggs is 10 μg / kg as specified in GB 31650 "National Food Safety Standard Maximum Residue Limits of 41 Veterinary Drugs in Food" (GB 31650.1-2022) issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

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

[0076] Quantitative determination

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

[0078] b. If the detection result exceeds the upper limit of the quantitative detection limit, dilute it with diluent and detect again.

[0079] Example Two: Preparation and use of a detection kit for sulfadimethoxine (SMZ) in chicken 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 of quantum dot fluorescent microspheres (10 mg / mL), surface carboxyl modified CdSe / ZnS quantum dot fluorescent microspheres (excitation 365 nm, emission 630 nm), and mix with 2 mL of MES buffer (0.05 mM, pH 6.0) after vortexing; sequentially add the crosslinking agent carbodiimide, N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared and used immediately), and react at room temperature for 15 minutes; centrifuge at 4°C for 20 minutes (centrifugal force 7000 g), remove the supernatant; add 2 mL of boric acid buffer (10 mM, pH 8.0) and vortex mix, and if necessary, perform ultrasonic treatment; then add 15 μg of anti-mouse IgG monoclonal antibody 1 and mix, and after reacting at room temperature for 2 hours, add 500 μL of 10% BSA. Mix, react at room temperature for 30 minutes; after centrifuging at 4°C for 30 minutes (centrifugal force 7000 g), remove the supernatant, and wash twice with 10 mM boric acid buffer (pH 8.0); add 300 μL of storage solution (2% sucrose, 1% alginic acid, 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, the coupled 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 30 g of CNBr activated Sepharose gel dry powder, suspend it in 250 mL of 1 mM hydrochloric acid solution, and after complete swelling, obtain 100 mL of sol; wash in a suction filter funnel with 250 mL of 1 mM hydrochloric acid, and then rinse 5 times with a binding solution containing 0.1 M sodium bicarbonate and 0.5 M sodium chloride (how much); then slowly stir the gel with an equal volume of 2 mg / mL SMZ-BSA at room temperature for 3 hours, and then slowly stir with 50 mg / mL bovine serum albumin (BSA) (how much) at room temperature for 1.5 hours; transfer to a suction filter funnel, and wash thoroughly with 5 times the volume of 0.01 M phosphate buffer solution (PBS), and the last time with 0.01 M PBS containing 0.3% sodium azide (NaN3), and after suctioning, pack into a conversion column, which is similar in size and shape to a 200 μL pipette tip, and the bottom is sealed with a plug and the top is sealed with a film.

[0086] 4. Preparation of general test strip

[0087] (1) Preparation of sample pad

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

[0089] (2) Preparation of conjugate release pad

[0090] Soak the polyester fiber membrane in 10 mM phosphate buffered saline (containing 0.1% Tween-20 and 1% BSA, pH 7.4) for 5 minutes, and dry at 37°C overnight.

[0091] Spray the prepared quantum dot fluorescent microsphere detection probe solution on the conjugate release pad at a rate of 10 μL / cm, dry in a vacuum drying oven at 37°C for 6 hours, and store under room temperature drying conditions.

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

[0093] Spray 2 mg / mL anti-mouse IgG monoclonal antibody 2 and 4 mg / mL Protein G at a rate of 0.75 μL / cm on the detection line position and the quality control line position, respectively, with a distance of 5 mm between the two lines, place the NC membrane in a constant temperature drying oven at 37°C for 12 hours, and store under room temperature drying conditions.

[0094] 5. Assembly of test strip

[0095] Adhere the NC membrane in the center of the plastic back plate, and adhere the sample pad, conjugate release pad, and filter paper in sequence at one end of the NC membrane, and adhere the water absorption paper at the other end. Then, cut into single strips with a width of 4 mm, seal in light-shielding bags, and store under room temperature drying conditions.

[0096] 6. Detection of SMZ in eggs

[0097] (1) After the egg sample is shelled, homogenize it at 500 r / min for 20 s to mix the egg white and yolk thoroughly; weigh 1.0 g of the homogenate into a centrifuge tube, add 5 mL of phosphate buffer, and shake gently by hand for 20 min to avoid emulsification; centrifuge at 4000 r / min for 10 min at room temperature, and detect the supernatant after diluting 4-fold with phosphate buffer.

[0098] (2) Add 200 μL of the above sample solution 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 open the sealing film on the top of the conversion column, pull out the small plug below, place a receiving dish underneath, pour in the mixed solution, about 1 minute (when the liquid no longer continuously drips), insert the test strip sample pad into the receiving dish, and after 10 minutes, realize the qualitative and quantitative detection of SMZ in eggs.

[0100] Qualitative determination

[0101] Use a 365nm LED lamp cylinder to irradiate the NC membrane of the test strip.

[0102] a. First, check if the C line is colored. If not, the test strip is invalid;

[0103] b. If the C line is normally colored, the result is determined according to Figure 5 .

[0104] Note: The maximum residue limit of SMZ in eggs is 10 μg / kg as specified in 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).

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

[0106] Quantitative determination

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

[0108] b. If the detection result exceeds the upper limit of the quantitative detection limit, dilute it with diluent and detect again.

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

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

[0111] Take 10 μL green AIE fluorescent microspheres (10 mg / mL; excitation 365 nm, emission 510 nm), vortex mixed with 2 mL MES buffer (0.05 mM, pH 6.0); then add crosslinking agent carbodiimide, N-hydroxysuccinimide (carbodiimide and N-hydroxysuccinimide are prepared and used immediately), room temperature reaction for 15 minutes; 4°C centrifugation for 20 minutes (centrifugal force 7000g), remove supernatant; add 2 mL boric acid buffer (10 mM, pH 8.0), vortex mix, if necessary, ultrasonic treatment; then add 15 μg anti-mouse IgG monoclonal antibody 1 mix, room temperature reaction for 2 hours, then add 500 μL of 10% BSA. Mix, room temperature reaction for 30 minutes; 4°C centrifugation for 30 minutes (centrifugal force is 7000g) after removing the supernatant, washed with 10 mM boric acid buffer (pH 8.0) twice; add 300 μL of storage solution (2% sucrose, 1% alginic acid, 1% polyethylene glycol 20000, 1% casein and 0.4% Tween-20) 4°C storage.

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

[0113] The coupling method is carbodiimide method, the coupling protein is bovine serum albumin (BSA), the coupling ratio is 1:60, and PROG-BSA is obtained after coupling and dialysis purification.

[0114] 3. Preparation of PROG conversion column

[0115] Take 30 g of CNBr activated Sepharose gel dry powder, suspend in 250 mL of 1 mM hydrochloric acid solution, and completely swell to obtain 100 mL of sol; wash in a suction filter funnel with 250 mL of 1 mM hydrochloric acid, and then rinse with 1 L of binding liquid containing 0.1 M sodium bicarbonate and 0.5 M sodium chloride for 5 times; then the gel is slowly stirred with equal volume of 2 mg / mL PROG-BSA at room temperature for 3 hours, and then 50 mg / mL equal volume of bovine serum albumin (BSA) is slowly stirred at room temperature for 1.5 hours; transfer to a suction filter funnel, and wash with 15 times volume of 0.01 M phosphate buffer solution (PBS) for 5 times, and the last time is washed with 0.01 M PBS containing 0.3% sodium azide (NaN3), and then suction dry and load into the conversion column, which is similar in size and shape to a 200 μL pipette tip, and the bottom end is sealed with a plug and the top end is sealed with a film.

[0116] 4. Preparation of general test strip

[0117] (1) Preparation of sample pad

[0118] 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 naturally air-dried at room temperature.

[0119] (2) Preparation of the conjugate release pad

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

[0121] The prepared AIE fluorescent microsphere detection probe solution was sprayed on the conjugate release pad at a rate of 10 μL / cm, dried in a vacuum drying oven at 37°C for 6 hours, and stored under room temperature drying conditions.

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

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

[0124] 5. Assembly of the test strip

[0125] The NC membrane was adhered in the center of the plastic back plate, and the sample pad, the conjugate release pad and the filter paper were sequentially adhered to one end of the NC membrane, and the water absorption paper was adhered to the other end. Subsequently, the test strip was cut into a single strip with a width of 4 mm, sealed in an opaque bag, and stored under room temperature drying conditions.

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

[0127] (1) The sample to be tested was added to the sample diluent (the main component was Tris-HCl) and mixed, and the pH was adjusted to the range of 6.5-8.0.

[0128] (2) 200 μL of the above sample solution was taken and added to a test tube containing 20 μg of PROG monoclonal antibody dry powder, and mixed.

[0129] (3) The conversion column was taken out and inserted into the environmentally friendly paper holder, the sealing film on the conversion column was torn off, the small plug below was pulled out, the bottom was placed on the receiving dish, the mixed solution was poured in, about 1 minute (when the liquid no longer continuously dripped), the test strip was inserted into the receiving dish, and after 10 minutes, the qualitative and quantitative detection of PROG in whole blood, serum and plasma samples was realized.

[0130] Qualitative determination

[0131] The test strip NC membrane is irradiated by a 365nm LED lamp barrel.

[0132] a. First, check whether the C line is colored, if not, the test strip is invalid;

[0133] b. If the C line is normally colored, the result is determined according to Figure 6 .

[0134] Note: The normal value of progesterone in different periods is as follows: 0.6-1.9nmol / L in the follicular phase, 2.4-9.4nmol / L in the ovulation phase, and 20.7-102.4nmol / L after ovulation.

[0135] Positive determination value: when the concentration of PROG is not less than 0.6nmol / L, the reagent kit can detect a positive result.

[0136] Quantitative determination

[0137] a. Open the test strip fluorescence signal analyzer, insert the detection card into the detection card slot at the lower right, and click the "test" on the instrument interface, so that the quantitative test result is automatically given by the instrument;

[0138] b. If the detection result exceeds the upper limit of the quantitative detection limit, the sample can be diluted with a diluent and then detected again.

[0139] In summary, the kit based on the double-antibody sandwich mode for detecting small molecules provided by the application can realize various output modes such as colorimetry and fluorescence, and can be applied to the sensitive detection of various small molecule targets.

Claims

1.A small molecule detection kit, mainly comprising a mouse IgG monoclonal antibody of a target small molecule, a conversion column, a universal test strip and an environmentally friendly paper support. The conversion column is filled after covalent coupling of the target small molecule or its artificial antigen with a gel having a supporting function, and has a sealing film at the top and a plug at the bottom. The target small molecule is enrofloxacin, sulfadimidine or progesterone. The gel having a supporting function comprises agarose or cellulose. The universal test strip comprises filter paper, a sample pad, a binding and releasing pad, an NC membrane and water-absorbing paper. wherein The binding and releasing pad is fixed with a probe having a signal output capability, i.e. a nano material 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 nano material having a signal output capability comprises colloidal gold or fluorescent microspheres. Anti-mouse IgG monoclonal antibody 1 and anti-mouse IgG monoclonal antibody 2 are a pair of matched antibodies for detecting IgG. 2.The small molecule detection kit according to claim 1, wherein the environmentally friendly paper support comprises two types: a foldable support or a pasted support, for vertical fixation of the conversion column when the kit is used. 3.The small molecule detection kit according to claim 1, wherein the conversion column is further provided with a receiving dish.

Images