WO2019024148A1 - 用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法 - Google Patents

用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法 Download PDF

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WO2019024148A1
WO2019024148A1 PCT/CN2017/098611 CN2017098611W WO2019024148A1 WO 2019024148 A1 WO2019024148 A1 WO 2019024148A1 CN 2017098611 W CN2017098611 W CN 2017098611W WO 2019024148 A1 WO2019024148 A1 WO 2019024148A1
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acetylcholinesterase
colloidal gold
monoclonal antibody
pad
test strip
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PCT/CN2017/098611
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English (en)
French (fr)
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眭涛
曹晓建
戈应滨
唐健
葛大伟
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南京医科大学第一附属医院
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Publication of WO2019024148A1 publication Critical patent/WO2019024148A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

  • the invention relates to the field of immunodiagnostic detection, in particular to a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase and a preparation method thereof.
  • peripheral nerve suture reconstruction technology The biggest problem with nerve suture is how to quickly and accurately identify the inner and inner bundles of the nerve trunk during surgery. At present, there is a lack of clinically accurate methods for accurately identifying the characteristics of peripheral nerve bundles in a short period of time. Once a mismatch of different types of nerve bundles occurs, the recovery rate of postoperative neurological functions will be seriously affected. Therefore, how to quickly and accurately identify the nature of peripheral nerve bundles has become a worldwide problem that microsurgery is extremely concerned about.
  • Immunochromatography is a fast and reliable diagnostic technique. The principle is to fix a specific antibody to a certain zone of the nitrocellulose membrane. When the dried nitrocellulose is immersed in the sample (urine, After serum and solution), the sample will move forward along the membrane due to capillary action. When moving to the region where the antibody is immobilized, the corresponding antigen in the sample will specifically bind to the antibody, if stained with immunocolloidal gold. This area can be displayed in a certain color to achieve a specific immunodiagnosis.
  • the method of curing cholinesterase antibody on the surface of the immunosensor reaction chip is adopted, and the sensitivity of the immunosensor is high, rapid, accurate and specific, and the content of cholinesterase in the solution is rapidly detected.
  • this method has been successfully used to identify the anterior and posterior roots of the spinal cord (1, Sui T, Ge Y, Liu W, Zhao ZK, Zhang N, Cao X .An acetylcholinesterase antibody-based quartz crystal microbalance for the rapid identification of spinal ventral and dorsal roots.PLoS One.2013 Jul 23;8(7):e69049.2, Sui T,Que J,Kong D,Xie H,Wang D , Shi K, Cao X, Li X. Rapid identification of spinal ventral and dorsal roots using a quartz crystal microbalance. Neural Regen Res. 2013 Mar 15; 8 (8): 686-92.
  • the stability of the chip is poor: the existing scheme is to first establish an inert hydrophobic substance on the surface of the crystal metal, mainly polyethyleneimine and 3-aminopropyl-triethoxysilane, and then use glutaraldehyde as the The cross-linking reagent acetylcholinesterase antibody binds. Glutaraldehyde is more chemically active, and once oxidized by air, its ability to bind antibodies is reduced. Affecting the binding of antigen-antibodies to the surface of the chip not only affects the sensitivity of the sensor, but also changes the result depending on the ability of the antibody to bind to the chip. The stability of the chip is poor, which directly limits its clinical application.
  • the convenience of the use of the chip is poor:
  • the chip is first activated before starting the test, then the glutaraldehyde is combined with the chip, and then the antibody is injected into the reaction cell of the sensor.
  • the operation is cumbersome, and the antibody needs to be refrigerated or cryopreserved. Improper storage of the antibody will also lower the potency and reduce the sensitivity of the sensor.
  • the principle underlying an immunosensor is an antigen-antibody reaction whose specificity depends on the specificity of the antibody.
  • the monoclonal antibody used in the above scheme has an antigen derived from a synthetic polypeptide, and the prepared antibody can recognize the whole protein of acetylcholinesterase, and the surface of human red blood cells also contains acetylcholinesterase.
  • the content of acetylcholinesterase in the nerve cross-section solution used for detection is extremely small (about 1 ⁇ g/ml), so blood contamination during surgery affects the reading of cholinesterase by the immunosensor, which inevitably affects the judgment of the result.
  • the immunosensor is based on Quartz Oscillation Balance Technology (QCM).
  • QCM Quartz Oscillation Balance Technology
  • the technical problem to be solved by the present invention is to provide a colloidal gold chromatography rapid diagnostic test strip for directly detecting acetylcholinesterase.
  • the nature of the nerve bundle is detected in real time.
  • the test strip can visually determine the content of acetylcholinesterase in the nerve bundle eluate, thereby quickly identifying the peripheral nerve motion beam and the sensory beam, and realizing the correct alignment of the motion beam to the motion beam and the sensory beam to the sensory beam.
  • the technical solution of the present invention provides a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase, which is provided on a hard plastic bottom plate. Connected absorbent filter paper, nitrocellulose membrane test pad, quality control zone, detection zone, colloidal gold pad and sample pad, commercially available rabbit anti-mouse IgG is fixed on the control area of the test pad, and the detection zone on the test pad is detected An anti-acetylcholinesterase monoclonal antibody is immobilized.
  • anti-acetylcholinesterase monoclonal antibody is a mouse anti-acetylcholinesterase antibody.
  • the above-mentioned quality control zone is sprayed with an anti-collection combined with a colloidal gold-labeled acetylcholinesterase monoclonal antibody body.
  • the above colloidal gold pad is sprayed with a colloidal gold-labeled acetylcholinesterase monoclonal antibody.
  • the colloidal gold-labeled anti-cholinesterase monoclonal antibody has a colloidal gold diameter of 20 nm to 100 nm and an anti-cholinesterase monoclonal antibody concentration of 45 ⁇ g to 100 ⁇ g/ml.
  • the colloidal gold-labeled anti-cholinesterase monoclonal antibody has a colloidal gold diameter of 40 nm to 100 nm and an anticholinesterase monoclonal antibody concentration of 64 ⁇ g to 100 ⁇ g/ml.
  • the colloidal gold-labeled anti-acetylcholinesterase monoclonal antibody is obtained by hybridizing the purified acetylcholinesterase monoclonal antibody with rat brain tissue to obtain a specific neurogenic acetylcholinesterase monoclonal antibody as a colloid An alternative to gold labeling; then the obtained monoclonal antibodies are separately HRP-labeled, and a pairwise pairwise pairing test is performed to determine an antibody pair that can be sandwiched to test standard acetylcholinesterase as a paired antibody, that is, a colloidal gold-labeled anti-acetylcholinesterase Monoclonal antibodies.
  • the invention also includes a method for preparing an acetylcholinesterase monoclonal antibody, comprising the steps of:
  • an acetylcholinesterase protein-antibody complex of a nervous tissue is obtained by immunoprecipitation using a monoclonal antibody of acetylcholinesterase and rat neural tissue;
  • mice Six female 6-week-old BALB/C mice were selected and immunized with the immunogen according to the time period. The whole cycle was 7 to 10 weeks, and the third boost was based on the specific experiment. Request treatment or continue to strengthen immunity;
  • the present invention also includes a method for preparing a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase, comprising the steps of:
  • Adhesive filter paper, nitrocellulose membrane test pad, colloidal gold pad (absorbent glass fiber) and sample pad (absorbent glass fiber) are adhered to the hard plastic substrate from top to bottom in order, and stored in vacuum dry state for use.
  • the anti-acetylcholine monoclonal antibody is labeled with colloidal gold of different diameters to prepare a colloidal gold pad, and the paired anti-acetylcholine monoclonal antibody is sprayed onto the detection area of the test pad, and the rabbit anti-mouse IgG is used. Spray Point to the quality control area of the test pad;
  • the invention adopts a monoclonal antibody of acetylcholinesterase, and extracts and purifies the neuro-acetylcholinesterase protein from the brain tissue of rats by immunoprecipitation, and obtains a monoclonal antibody of neuro-acetylcholinesterase by immunizing the mouse.
  • a paired monoclonal antibody that specifically recognizes a high-affinity acetylcholinesterase expressed in neural tissue that does not cross-react with acetylcholinesterase on the erythrocyte membrane is negatively selected from the red blood cell suspension, and is prepared using the antibody.
  • the colloidal gold test strip is used to detect the acetylcholinesterase content of the nerve tissue at the end, showing good sensitivity and specificity.
  • the invention aims to identify the need for the sensory branch and the motion branch in the nerve bundle during the operation, and adjusts the colloidal gold test strip by adjusting the diameter of the colloidal gold, the concentration of the antibody bound to the colloidal gold, and the method of adjusting the pore size of the nitrocellulose membrane. Sensitivity, so that the sensory branch (negative) and the exercise branch (strong positive) can be quickly and accurately distinguished to meet clinical needs.
  • the colloidal gold test strip of the present invention is used for visually detecting acetylcholinesterase in an eluate, and has the following advantages: 1
  • the reagent and the sample amount are extremely small, and the sample amount can be as low as 10-20 ⁇ l, thereby increasing the eluent.
  • the concentration of acetylcholinesterase is not easy to produce false negative results; 2 no need for expensive instruments, more suitable for field application; 3 the detection speed of the invention is fast, generally 5-10 minutes can produce results; 4 test strips can be stored at room temperature, Just keep the test strip dry; 5 high sensitivity, can reach the level of ELISA (pg / ml), and combined with silver staining, the sensitivity of the detection is greater and greatly improved, fully meet the detection of acetylcholine in the nerve bundle eluate The need for esterase content.
  • the rapid nature of the test strip is derived from the inherent properties of colloidal gold immunochromatography and is closely related to the choice of raw material, especially the pore size of the NC membrane. The accuracy depends on the specificity of the anti-cholinesterase monoclonal antibody.
  • Figure 1 is a schematic view of the structure of the present invention. Among them: 1, water-absorbing filter paper, 2, nitrocellulose membrane test pad, 3, colloidal gold-labeled acetylcholinesterase monoclonal antibody colloidal gold pad, 4, sample pad, 5, fixed on nitrocellulose membrane for quality Controlled commercially available rabbit anti-mouse IgG, 6, acetylcholinesterase monoclonal antibody immobilized on a nitrocellulose membrane for detecting samples, 7. Hard plastic substrate.
  • Figure 2 is a diagram of co-immunoprecipitation of rat brain tissue.
  • Lane 1 293T cell lysate transfected with acetylcholinesterase;
  • Lane 2 rat brain homogenate. The above two lanes were positive as Western blot analysis, indicating that the 293T cell lysate transfected with acetylcholinesterase and the rat brain homogenate contained acetylcholinesterase protein.
  • Lane 3 is a protein sample of the monoclonal antibody of clone No. AP0961 co-immunoprecipitated with brain tissue homogenate, Western blot analysis showed that the monoclonal antibody of clone No.
  • Lane 4 a protein sample of rat IgG and brain tissue homogenate immunoprecipitated, negative by Western blot, indicating that rat IgG could not recognize acetylcholinesterase in brain tissue.
  • Lane 5 is a protein sample of the monoclonal antibody of clone No. 53E11c9 co-immunoprecipitated with brain tissue homogenate, and positive by Western blot, indicating that the monoclonal antibody of clone No. 53E11c9 can recognize acetylcholinesterase in brain tissue.
  • Lane 6 is a protein sample of the monoclonal antibody of clone No.
  • Lane 7 is a protein sample of rat IgG and brain tissue homogenate immunoprecipitated, negative by Western blot, indicating that rat IgG can not recognize acetylcholinesterase in brain tissue.
  • Lane 8 is a protein sample of the monoclonal antibody of clone No. 1H1H4 co-immunoprecipitated with brain tissue homogenate, and positive by Western blot, indicating that the monoclonal antibody of clone No.
  • Lane 9 is a protein sample of rat IgG and brain tissue homogenate immunoprecipitated, negative by Western blot, indicating that rat IgG can not recognize acetylcholinesterase in brain tissue.
  • Lane 10 a protein sample of the monoclonal antibody of clone No. AP0962 co-immunoprecipitated with brain tissue homogenate, which was negative by Western blot, indicating that the monoclonal antibody of clone No. AP0962 could not recognize the acetylcholinesterase in brain tissue.
  • Lane 11 is a protein sample of the monoclonal antibody of clone No.
  • Lane 12 a protein sample immunoprecipitated by polyclonal antibody and brain tissue homogenate, positive by Western blot, indicating that the polyclonal antibody can recognize acetylcholinesterase in brain tissue.
  • Figure 3 is a Western blot of a monoclonal antibody prepared in accordance with the procedure of Example 1 and a rat brain homogenate; wherein 3 is a protein Marker. A positive band was observed in lane 28, and no significant positive reaction was observed in the other lanes. Therefore, antibody No. 28 (clone number: 2H12-F-7) was selected for the selection of colloidal gold test strips;
  • Figure 4 is a Western blot of a monoclonal antibody prepared in accordance with the procedure of Example 1 and a rat brain homogenate, wherein 4 is a protein Marker. Positive bands were observed in lanes 41 and 42, and no significant positive reaction was observed in the other lanes. Therefore, antibody No. 41 (clone number: 1F4-C4-F2-D5) and No. 42 (clone number: 3A11-C5) were selected for colloidal gold.
  • Colloidal gold pad has a colloidal gold diameter of 40 nm, a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 64 ⁇ g/ml; and a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml. When the concentration of acetylcholinesterase is 1 ⁇ g/ml, the red wine band visible at the same time in the detection zone and the quality control zone of the colloidal gold test strip is a positive result;
  • Colloidal gold pad has a colloidal gold diameter of 20 nm, a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 58 ⁇ g/ml; and a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml. When the concentration of acetylcholinesterase is 1 ⁇ g/ml, only the burgundy band visible in the macroscopic control strip of the colloidal gold test strip has a negative result;
  • Colloidal gold pad has a colloidal gold diameter of 100 nm, a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 45 ⁇ g/ml; and a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml. When the concentration of acetylcholinesterase is 1 ⁇ g/ml, only the wine-red band visible in the control area of the colloidal gold test strip has a negative result.
  • FIG. 8 Colloidal gold pad has a colloidal gold diameter of 40 nm, a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 64 ⁇ g/ml; and a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml.
  • Colloidal gold pad has a colloidal gold diameter of 40 nm, a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 64 ⁇ g/ml; and a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml. When the root supernatant was added to the spinal cord, only the wine-red band visible in the quality control area of the colloidal gold test strip showed a negative result.
  • a monoclonal antibody (clone number: 2H12-F-7) bound to colloidal gold is 64 ⁇ g/ml
  • a nitrocellulose membrane detection zone 6 is a paired monoclonal antibody. (Clone number: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml.
  • mice Six female 6-week-old BALB/C mice were selected and basal and boosted with the immunogen on a time period (7 to 10 weeks), as shown in Table 1. After the third boost, follow the specific experimental requirements or continue to strengthen the immunization.
  • Preparation of ascites 2-3 weeks before the planned injection of cells, select Balb/c mice (must be healthy) pre-stimulate with 500 ⁇ l of liquid paraffin, take about 1 ⁇ 10 6 cells for injection, and inject 500 ⁇ l per mouse. . 3-4 per hybridoma can be used to ensure that the amount of ascites is above 20ml.
  • Antibody purification The supernatant or ascites of the cells is separated and purified by an immunogen (acetylcholinesterase protein) affinity column, and the receiving solution obtained by the purification process is dialyzed, concentrated and stored.
  • immunogen acetylcholinesterase protein
  • the obtained monoclonal antibody was hybridized with rat brain tissue by western blot, and a specific monoclonal antibody was selected as an alternative to the colloidal gold test strip.
  • a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase comprising a water-absorbing filter paper 1 and a nitrocellulose membrane connected in series from the top to the bottom on a hard plastic bottom plate 7 Millipore, 135S, pore size 8 ⁇ m) test pad 2 and colloidal gold pad (absorbent glass fiber) 3 and sample pad (40 ⁇ m colloidal gold-labeled acetylcholinesterase monoclonal antibody (clone: 2H12-F-7) Water-absorbent glass fiber 4, a commercially available rabbit anti-mouse IgG (Abcam ab46540) was immobilized on the control pad 5 on the test pad, and the acetylcholinesterase monoclonal antibody was immobilized in the detection zone 6 on the test pad (clone: 1F4-C4-F2) -D5).
  • the test strip assembly method the water absorbing filter paper 1, the test pad 2 and the sample pad 4 are respectively assembled on the plastic bottom plate according to the figure, and the combination of the accessory and the plastic bottom plate can be bonded by the viscous material.
  • the assembled cardboard is cut longitudinally and cut into strips having a width of 4 mm.
  • the colloidal gold pad 3 is inserted between the sample pad 4 and the test pad 2 to be a test strip for cholinesterase detection.
  • the anti-acetylcholine monoclonal antibody is labeled with colloidal gold of different diameters to form a colloidal gold pad, and the paired anti-acetylcholine monoclonal antibody is sprayed to the detection zone 6 of the test pad.
  • the colloidal gold pad 3 is inserted between the sample pad 4 and the test pad 2, and the standard acetylcholinesterase PBST solution is dropped onto the sample pad, and the sample is run for 10 minutes, and the sandwich which can be used for detecting the standard acetylcholinesterase is selected according to the detection region phenomenon.
  • Monoclonal antibody combination Monoclonal antibody combination.
  • the colloidal gold pad of the present embodiment has a colloidal gold diameter of 40 nm, a monoclonal antibody (clone number: 2H12-F-7) combined with colloidal gold has a concentration of 64 ⁇ g/ml, and a detection zone 6 on the nitrocellulose membrane is a paired single.
  • the concentration of the cloned antibody (clone: 1F4-C4-F2-D5) was 100 ⁇ g/ml.
  • Different concentrations of acetylcholinesterase PBST solution concentration of 1ng/ml, 10ng/ml, 100ng/ml, 1 ⁇ g/ml, 10 ⁇ g/ml
  • Two obvious red lines appeared in the area 6 and the quality control area 5 and a red line appeared in the quality control area 5 as a negative result.
  • the acetylcholinesterase monoclonal antibody and the cholinesterase solution of the present embodiment have good adaptability and are stored in a buffer capable of effectively maintaining antibody activity and eliminating false positives.
  • the buffer formulation 0.01 M PBS PH7.2 + 0.5% cold fish skin gelatin (cold water fish skin collagen, purchased from Sigma) + 0.05% sodium azide (sodium azide).
  • Preparation method Take 100ml of 0.01M PBS, add 50mg of cold water fish skin collagen, stir and dissolve, heat and boil at 1.2 atmospheres for 20 minutes, cool to room temperature, add sodium azide 200mg, filter, and store in separate parts.
  • the specific method is the same as that of Example 1 and Example 2, except that the colloidal gold pad used has a colloidal gold diameter of 20 nm, and the colloidal gold-binding monoclonal antibody (clone: 2H12-F-7) has a concentration of 58 ⁇ g/ml.
  • the detection zone 6 on the nitrocellulose membrane is a paired monoclonal antibody (clone: 1F4-C4-F2-D5).
  • Different concentrations of acetylcholinesterase PBST solution concentration of 1ng/ml, 10ng/ml, 100ng/ml, 1 ⁇ g/ml, 10 ⁇ g/ml
  • Two obvious red lines appeared in the area 6 and the quality control area 5, and only one red line appeared in the quality control area.
  • the specific method is the same as that of Example 1 and Example 2, except that the colloidal gold pad used has a colloidal gold diameter of 100 nm, and the colloidal gold-binding monoclonal antibody (clone number: 2H12-F-7) has a concentration of 45 ⁇ g/ml.
  • the detection zone 6 on the nitrocellulose membrane was a paired monoclonal antibody (clone: 1F4-C4-F2-D5) at a concentration of 100 ⁇ g/ml.
  • Different concentrations of acetylcholinesterase PBST solution concentration of 1ng/ml, 10ng/ml, 100ng/ml, 1 ⁇ g/ml, 10 ⁇ g/ml
  • Zone 6 and QC zone 5 appear two A positive red line is a positive result, and only one red line appears in the quality control area 5 to be negative.
  • the present invention selects a colloidal gold diameter of 40 nm, a monoclonal antibody (clone number: 2H12-F-7) concentration of 64 ⁇ g/ml, and a monoclonal antibody (clone number: 1F4-C4-F2-D5) concentration of 100 ⁇ g/ml. .
  • Example 2 Cut a segment of human anterior root of about 1 mm, place it in 250 ⁇ l of PBS solution, mix by shaking and centrifuge to obtain the supernatant, and then apply 100 ⁇ l of supernatant supernatant to the colloidal gold test strip obtained in Example 2. On the sample pad, the sample was run for 10 minutes, and a typical positive result appeared on the test strip (see Figure 8). The root poster of the human spinal cord was also taken, and the supernatant was applied to the colloidal gold test strip obtained in Example 2. On the sample pad, run the sample for 10 minutes and the test strip is negative (see Figure 9). According to the above results, the colloidal gold test strip obtained in Example 2 can identify the anterior root and the posterior root of the spinal cord.
  • the concentration of acetylcholinesterase in the anterior root solution of the spinal cord was 2.98 ⁇ g/ml
  • the concentration of acetylcholinesterase in the root solution of the spinal cord was 0.90 ⁇ g/ml (Sui T1, Ge Y, Liu W,Zhao ZK,Zhang N,Cao X.An acetylcholinesterase antibody-based quartz crystal microbalance for the rapid identification of spinal ventral and dorsal roots.PLoS One.2013Jul 23;8(7):e69049.). Therefore, the detection sensitivity of the colloidal gold test strip of the present invention to acetylcholinesterase is >0.90 ⁇ g/ml.

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Abstract

一种用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法，所述诊断试纸条包括设在硬质塑料底板上依次相连的吸水滤纸、固定有抗乙酰胆碱酯酶单克隆抗体（检测区）与兔抗小鼠IgG（质控区）的醋酸纤维膜的检测垫、标记有抗乙酰胆碱酯酶单克隆抗体的胶体金垫和放置样品的样品垫。胶体金试纸条用于肉眼检测溶液中的乙酰胆碱酯酶，具有以下优点：①试剂和样本用量极小，样本量可低至10～20μl，因而可以增加标本洗脱液中乙酰胆碱酯酶的浓度，不易出现假阴性结果；②不需贵重仪器，更适于现场应用；③时间大大缩短，提高了检测速度；④试纸条可在室温保存，只需保持试纸条干燥即可。

Description

用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法 技术领域
本发明涉及免疫诊断学检测领域，具体涉及用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法。
背景技术
随着社会工业的发展，周围神经损伤的发病率逐年上升。目前主要的治疗方式为周围神经缝合重建技术。而神经缝合最大的难题在于如何在手术中快速准确鉴别神经干内运动束和感觉束。目前临床上尚缺乏能在短时间内准确鉴别周围神经束性质的方法，一旦发生不同性质神经束的错误吻合必将严重影响术后神经功能的恢复率。因此如何快速准确鉴别周围神经束的性质已成为当今显微外科极其关注的世界难题。
免疫层析法(immunochromatography)是一种快速可靠的诊断技术，其原理是将特异的抗体先固定于硝酸纤维素膜的某一区带，当该干燥的硝酸纤维素一端浸入样品(尿液、血清和溶液)后，由于毛细管作用，样品将沿着该膜向前移动，当移动至固定有抗体的区域时，样品中相应的抗原即与该抗体发生特异性结合，若用免疫胶体金染色可使该区域显示一定的颜色，从而实现特异性的免疫诊断。
目前，采用在免疫传感器反应芯片表面固化胆碱酯酶抗体的方法，利用免疫传感器灵敏度高、快速、准确、特异性强的特点，快速检测溶液中胆碱酯酶的含量。根据运动神经的乙酰胆碱酯酶含量远高于感觉神经的事实，此方法已经成功用于鉴别脊髓的前根和后根(1、Sui T,Ge Y,Liu W,Zhao ZK,Zhang N,Cao X.An acetylcholinesterase antibody-based quartz crystal microbalance for the rapid identification of spinal ventral and dorsal roots.PLoS One.2013 Jul 23；8(7):e69049.2、Sui T,Que J,Kong D,Xie H,Wang D,Shi K,Cao X,Li X.Rapid identification of spinal ventral and dorsal roots using a quartz crystal microbalance.Neural Regen Res.2013 Mar 15；8(8):686-92.)本方法的建立为临床快速鉴别诊断周围神经感觉与运动支提供了可能。
但是此方案尚存在下列不足，影响其在临床的推广使用：
1、芯片稳定性较差：已有方案是首先在晶体金属表面建立一层惰性疏水物质，主要是聚乙烯亚胺和3-氨丙基-三乙氧基硅烷，然后再用戊二醛作为交联试剂乙酰胆碱酯酶抗体结合。戊二醛化学性质较为活泼，一旦被空气氧化，其结合抗体的能力会降低， 影响抗原抗体在芯片表面的结合，不但会影响传感器的敏感性，也会使结果随抗体与芯片结合的能力不同而发生改变。芯片的稳定性较差，直接限制其在临床的应用。
2、芯片使用的便利性较差：现有方案中，开始检测前首先是将芯片激活，再将戊二醛与芯片结合，再将抗体注射至传感器的反应池中。操作较为繁琐，且抗体需要冷藏或冷冻保存，抗体保存不当也会使其效价降低，传感器的敏感度降低。
3、方案中所使用的设备昂贵，每台设备约需人民币50万元，即使简化配置，也需人民币25万元以上。此外此方案操作繁琐，操作人员需要较为熟练的技术。
4、干扰因素较多，影响结果的判读。免疫传感器所依据的原理是抗原抗体反应，其特异性取决于抗体的特异性。上述方案所使用的单克隆抗体，其抗原来源于合成多肽，所制备的抗体可以识别乙酰胆碱酯酶的全蛋白，包括人红细胞表面也含有乙酰胆碱酯酶。用于检测的神经断面溶液中乙酰胆碱酯酶的含量极微(1μg/ml左右)，所以手术中的血液污染会影响免疫传感器对胆碱酯酶的读取，不可避免地会影响结果的判断。
5、免疫传感器是基于石英震荡天平技术(QCM)，QCM在液态环境中检测得到的芯片的频率变化值(即QCM信号)是由芯片表面质量改变与溶液粘弹性改变共同引起的，数据分析的复杂性显然易见。排除溶液粘弹性因素的影响很困难。
发明内容
发明目的：为了克服现有免疫传感器对神经束性质进行判断方法的不足，本发明所要解决的技术问题是提供了一种直接用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，用于实时检测神经束的性质。该试纸条可以通过肉眼判断神经束洗脱液中的乙酰胆碱酯酶的含量，从而快速鉴别出周围神经运动束与感觉束，实现运动束对运动束、感觉束对感觉束正确吻合。
技术方案：为了解决上述问题，本发明的技术方案是提供了一种用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，所述诊断试纸条包括设在硬质塑料底板上依次相连的吸水滤纸、硝酸纤维素膜的检测垫、质控区、检测区、胶体金垫以及样品垫，所述检测垫上的质控区上固定有市售兔抗鼠IgG，检测垫上的检测区固定有抗乙酰胆碱酯酶单克隆抗体。
其中，上述抗乙酰胆碱酯酶单克隆抗体为小鼠抗乙酰胆碱酯酶抗体。
其中，上述质控区喷涂有能与胶体金标记的乙酰胆碱酯酶单克隆抗体相结合的抗 体。
其中，上述胶体金垫喷涂有胶体金标记的乙酰胆碱酯酶单克隆抗体。
其中，上述胶体金标记的抗胆碱酯酶单克隆抗体的胶体金直径为20nm～100nm、抗胆碱酯酶单克隆抗体浓度为45μg～100μg/ml。
进一步地，作为优选，上述胶体金标记的抗胆碱酯酶单克隆抗体的胶体金直径为40nm～100nm、抗胆碱酯酶单克隆抗体浓度为64μg～100μg/ml。
其中，上述胶体金标记的抗乙酰胆碱酯酶单克隆抗体是通过将纯化后的乙酰胆碱酯酶单克隆抗体与大鼠脑组织进行杂交，进而获取特异性神经源性的乙酰胆碱酯酶单克隆抗体作为胶体金标记的备选；然后将获取的单克隆抗体分别进行HRP标记，进行两两组合配对检测，确定可夹心检测标准乙酰胆碱酯酶的抗体对作为配对抗体，即得胶体金标记的抗乙酰胆碱酯酶单克隆抗体。
本发明内容还包括乙酰胆碱酯酶单克隆抗体的制备方法，包括以下步骤：
1)免疫原的获得：利用乙酰胆碱酯酶单克隆抗体与大鼠神经组织通过免疫共沉淀法，得到神经组织的乙酰胆碱酯酶蛋白与抗体复合物；
2)将乙酰胆碱酯酶蛋白与抗体复合物用甘氨酸溶液解离下来，透析除去甘氨酸得到乙酰胆碱酯酶蛋白，作为免疫原；
3)小鼠免疫：挑选六只雌性6周龄的BALB/C小鼠，用免疫原按时间周期进行基础免疫和加强免疫，整个周期为7到10周，第3次加强免疫后根据具体实验要求进行处理或者继续加强免疫；
4)融合：在第二次加强免疫7天后和第三次加强免疫7天后分别进行阳性血的采集，进行ELISA检测效价，根据ELISA测定的小鼠血清稀释到1：10000时OD值需达到0.5以上，加强免疫三天后的小鼠，拉颈猝死后，取出脾脏研磨后，按常规方法进行融合，筛选的阳性孔进行多次亚克隆，直至100％阳性，即得到单克隆抗体。
本发明内容还包括用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条的制备方法，包括以下步骤：
1)在硬质塑料底板自上而下依次黏贴吸水滤纸、硝酸纤维素膜的检测垫，胶体金垫(吸水玻璃纤维)和样品垫(吸水玻璃纤维)，真空干燥状态下保存，备用。
2)按照试纸条组装步骤，分别将抗乙酰胆碱单克隆抗体标记不同直径的胶体金制成胶体金垫，能配对的抗乙酰胆碱单克隆抗体喷点到检测垫的检测区，将兔抗鼠IgG喷 点到检测垫的质控区；
3)将胶体金垫插入样品垫和检测垫之间，将标准乙酰胆碱酯酶的PBST溶液滴涂到样品垫上，跑样10分钟，根据检测区域现象筛选出可用于检测标准乙酰胆碱酯酶的夹心式单克隆抗体组合，当检测区出现清晰可见的酒红色条带，即为阳性结果。
本发明采用乙酰胆碱酯酶单克隆抗体，用免疫共沉淀的方法，从大鼠脑组织中分离提纯出神经型乙酰胆碱酯酶蛋白，免疫小鼠获得神经型乙酰胆碱酯酶单克隆抗体。以红细胞悬液负向筛选出与红细胞膜上的乙酰胆碱酯酶不发生交叉反应的、特异性识别表达于神经组织的、高亲和性的乙酰胆碱酯酶的配对单克隆抗体，用此抗体制备出胶体金试纸条，用以检测神经组织断端的乙酰胆碱酯酶含量，显示出较好的灵敏度与特异性。
本发明针对术中鉴别神经束中感觉支和运动支的需要，通过调整胶体金直径、与胶体金结合的抗体浓度、调整硝酸纤维素膜孔径的方法，合理设定了胶体金试纸条的敏感性，从而可以快速准确地分辨出感觉支(阴性)和运动支(强阳性)，达到临床需求。
有益效果：本发明胶体金试纸条用于肉眼检测洗脱液中的乙酰胆碱酯酶，具有以下优点：①试剂和样本用量极小，样本量可低至10～20μl，因而可以增加洗脱液中乙酰胆碱酯酶的浓度，不易出现假阴性结果；②不需贵重仪器，更适于现场应用；③本发明检测速度快，一般5-10分钟可出结果；④试纸条可在室温保存，只需保持试纸条干燥即可；⑤灵敏度高，可达到ELISA的水平(pg/ml)，而结合银染色时，检测的敏感性更大大提高，完全能满足检测神经束洗脱液中乙酰胆碱酯酶含量的需要。试纸条的快速特性来源于胶体金免疫层析法的固有特性，也与原材料选择特别是NC膜的孔径大小密切相关，准确性取决于抗胆碱酯酶单克隆抗体的特异性。
附图说明
图1为本发明的结构示意图。其中：1、吸水滤纸，2、硝酸纤维素膜检测垫，3、胶体金标记的乙酰胆碱酯酶单克隆抗体的胶体金垫，4、样品垫，5、固定于硝酸纤维素膜上用于质量控制的市售兔抗鼠IgG，6、固定于硝酸纤维素膜上用于检测样品的乙酰胆碱酯酶单克隆抗体，7、硬质塑料底板。
图2为大鼠脑组织的免疫共沉淀图。泳道1：转染乙酰胆碱酯酶的293T细胞裂解液；泳道2：大鼠脑组织匀浆液。上述两个泳道作为Input对照Western blot检测阳性，说明转染乙酰胆碱酯酶的293T细胞裂解液和大鼠脑组织匀浆液中含有乙酰胆碱酯酶蛋白。泳道3：是克隆号AP0961的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品， Western blot检测阳性，说明克隆号AP0961的单克隆抗体能够识别脑组织中的乙酰胆碱酯酶。泳道4：是大鼠IgG与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阴性，说明大鼠IgG不能识别脑组织中的乙酰胆碱酯酶。泳道5：是克隆号53E11c9的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阳性，说明克隆号53E11c9的单克隆抗体能够识别脑组织中的乙酰胆碱酯酶。泳道6：是克隆号62D12H4的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阳性，说明克隆号62D12H4的单克隆抗体能够识别脑组织中的乙酰胆碱酯酶。泳道7：是大鼠IgG与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阴性，说明大鼠IgG不能识别脑组织中的乙酰胆碱酯酶。泳道8：是克隆号1H1H4的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阳性，说明克隆号1H1H4的单克隆抗体能够识别脑组织中的乙酰胆碱酯酶。泳道9：是大鼠IgG与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阴性，说明大鼠IgG不能识别脑组织中的乙酰胆碱酯酶。泳道10：是克隆号AP0962的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阴性，说明克隆号AP0962的单克隆抗体不能识别脑组织中的乙酰胆碱酯酶。泳道11：是克隆号AP0962的单克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阴性，说明克隆号AP0962的单克隆抗体不能识别脑组织中的乙酰胆碱酯酶。泳道12：是多克隆抗体与脑组织匀浆免疫共沉淀的蛋白样品，Western blot检测阳性，说明多克隆抗体能够识别脑组织中的乙酰胆碱酯酶。
图3按照实施例1步骤制备的单克隆抗体与大鼠脑组织匀浆的Western blot；其中3，为蛋白Marker。泳道28可见阳性条带，其余泳道未见明显阳性反应，故选取抗体编号28号(克隆号：2H12-F-7)应用于胶体金试纸条的备选；
图4按照实施例1步骤制备的单克隆抗体与大鼠脑组织匀浆的Western blot，其中4为蛋白Marker。泳道41和42可见阳性条带，其余泳道未见明显阳性反应，故选取抗体编号41号(克隆号：1F4-C4-F2-D5)和42号(克隆号：3A11-C5)应用于胶体金试纸条的备选；
图5胶体金垫的胶体金直径为40nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)，浓度为100μg/ml。当乙酰胆碱酯酶浓度为1μg/ml时，胶体金试纸条的检测区与质控区同时出现肉眼可见的酒红色条带，即为阳性结果；
图6胶体金垫的胶体金直径为20nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为58μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)，浓度为100μg/ml。当乙酰胆碱酯酶浓度为1μg/ml时，只有胶体金试纸条的质控区出现肉眼可见的酒红色条带，即为阴性结果；
图7胶体金垫的胶体金直径为100nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为45μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)，浓度为100μg/ml。当乙酰胆碱酯酶浓度为1μg/ml时，只有胶体金试纸条的质控区出现肉眼可见的酒红色条带，即为阴性结果
图8胶体金垫的胶体金直径为40nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)，浓度为100μg/ml。当加入脊髓前根上清液时，胶体金试纸条的检测区与质控区同时出现肉眼可见的酒红色条带，即为阳性结果，且检测区颜色较图5更深
图9胶体金垫的胶体金直径为40nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)，浓度为100μg/ml。当加入脊髓后根上清液，只有胶体金试纸条的质控区出现肉眼可见的酒红色条带，即为阴性结果。
具体实施方式
下面结合附图对本发明作更进一步的说明。
实施例1：抗乙酰胆碱酯酶的单克隆抗体的制备
1、免疫原的获得
1)利用本课题组先期获取的单克隆抗体(发明授权号：ZL2012 1 0516 218.3；按照该专利中的方法制备得到单抗克隆号为AP0691、AP0962、53E11c9，62D12H4，1H1H4的单克隆抗体)，分别将500μl大鼠神经组织(脑组织)匀浆液(3μg/μl)中加入20μl浓度为1mg/ml的单克隆抗体(克隆号：AP0961，53E11c9，62D12H4，1H1H4，AP0962)与50μlAnti-mouse IgG(H+L)，F(ab')2 Fragment(
Figure PCTCN2017098611-appb-000001
Bead Conjugate)(Cell Signaling Technology公司，货号：5946)、20μl浓度为1mg/ml的自制兔抗乙酰胆碱酯酶多克隆抗体与50μlProtein G Agarose Beads(Cell Signaling Technology公司，货号37478)、50μlMouse IgG(
Figure PCTCN2017098611-appb-000002
Bead Conjugate)(Cell Signaling Technology公司，货号3420)，4℃摇床过夜。4℃12000g离心15分钟，弃去上清，得到乙酰胆碱酯酶单 克隆抗体与大鼠脑组织的免疫共沉淀样品。样品PBST清洗3次后，加入5X上样缓冲液(碧云天生物技术，货号P0015)煮沸后离心，取15μl上清与15μl转染乙酰胆碱酯酶的293T细胞裂解液和15μl鼠脑组织匀浆液一起SDS-PAGE电泳后转膜，进行常规Western blot，市售商品化乙酰胆碱酯酶抗体(Abcam，货号：ab70219)作为检测抗体；结果参见图2；根据以上结果，我们选取克隆号AP0961的单克隆抗体与鼠脑组织进行免疫共沉淀以获取用于制备胶体金试纸条的单克隆抗体的抗原。
2)将乙酰胆碱酯酶单克隆抗体AP0961与脑组织的免疫共沉淀样品用0.1M的甘氨酸溶液解离下来，透析除去甘氨酸得到乙酰胆碱酯酶蛋白，作为免疫原；
2、小鼠免疫
挑选六只雌性6周龄的BALB/C小鼠，用免疫原按时间周期进行基础免疫和加强免疫(整个周期为7到10周)，如表1。第3次加强免疫后根据具体实验要求进行处理或者继续加强免疫。
表1
Figure PCTCN2017098611-appb-000003
3、融合
在第二次加强免疫7天后和第三次加强免疫7天后分别进行阳性血的采集，进行ELISA检测效价。根据ELISA测定的小鼠血清稀释到1：10000时OD值需达到0.5以上，加强免疫三天后的小鼠，拉颈猝死后，取出脾脏研磨后，按常规方法进行融合，多次亚克隆，直至100％阳性，我们认为即得到单克隆株。
4、腹水制备及抗体纯化
腹水制备：计划注射细胞前2-3周，选Balb/c小鼠(小鼠一定要健康)用500μl液 体石蜡进行预刺激，取约1×106个细胞进行注射，每只老鼠注射500μl左右。每株杂交瘤打3-4只，可保证腹水量在20ml以上。
抗体纯化：用免疫原(乙酰胆碱酯酶蛋白)亲和柱对细胞上清或腹水进行分离纯化，纯化过程所得的接收液进行透析，浓缩并保存。
5、抗体的筛选：
采用western blot的方法将获得的单克隆抗体与大鼠脑组织进行杂交，选取特异性的单克隆抗体作为胶体金试纸条的备选。
根据结果，参见图3的28号(克隆号：2H12-F-7)，图4的41号(克隆号：1F4-C4-F2-D5)，42号(克隆号：3A11-C5)被应用于胶体金试纸条的备选。
6、抗体标记及夹心抗体配对
将3个单克隆抗体分别进行HRP标记，进行两两组合配对检测，确定可夹心检测标准乙酰胆碱酯酶的抗体对。最终选取了2H12-F-7与1F4-C4-F2-D5，作为配对抗体即为胶体金标记的抗乙酰胆碱酯酶单克隆抗体。
实施例2：检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条的制备
一种用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，所述诊断试纸条包括设在硬质塑料底板7上从上到下依次相连的吸水滤纸1、硝酸纤维素膜(Millipore公司，135S，孔径8μm)的检测垫2和滴有40nm胶体金标记的乙酰胆碱酯酶单克隆抗体(克隆号：2H12-F-7)的胶体金垫(吸水玻璃纤维)3以及样品垫(吸水玻璃纤维)4，检测垫上的质控区5固定有市售兔抗鼠IgG(Abcam公司ab46540)，检测垫上的检测区6固定有乙酰胆碱酯酶单克隆抗体(克隆号：1F4-C4-F2-D5)。
试纸条的装配方法：在塑料底板上分别将吸水滤纸1、检测垫2以及样品垫4按图装配，配件与塑料底板的结合可用粘性材料粘接。装配好的纸板按纵向剪切，裁成宽度为4mm的条状，将胶体金垫3插入样品垫4与检测垫2之间即为胆碱酯酶检测用试纸条。按照试纸条组装步骤，分别将抗乙酰胆碱单克隆抗体标记不同直径的胶体金制成胶体金垫，能配对的抗乙酰胆碱单克隆抗体喷点到检测垫的检测区6。将胶体金垫3插入样品垫4和检测垫2之间，将标准乙酰胆碱酯酶的PBST溶液滴涂到样品垫上，跑样10分钟，根据检测区域现象筛选出可用于检测标准乙酰胆碱酯酶的夹心式单克隆抗体组合。
本实施中的胶体金垫的胶体金直径为40nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)浓度为100μg/ml。将不同浓度的乙酰胆碱酯酶PBST溶液(浓度分别为1ng/ml、10ng/ml、100ng/ml、1μg/ml、10μg/ml)，滴涂到样品垫上，跑样10分钟，肉眼观察，在检测区6和质控区5出现两条明显红线的为阳性结果，只在质控区5出现一条红线则为阴性结果。
本实施例中，当乙酰胆碱酯酶浓度为1μg/ml时，出现典型阳性结果(见图5)。
本实施例的乙酰胆碱酯酶单克隆抗体和胆碱酯酶溶液都有良好的适应性，保藏于能有效保持抗体活性和消除假阳性的缓冲液中。
该缓冲液配方：0.01M PBS PH7.2+0.5％cold fish skin gelatin(冷水鱼皮肤胶原，购于Sigma公司)+0.05％sodium azide(叠氮钠)。
制备方法：取0.01M PBS 100ml，再加入冷水鱼皮肤胶原50mg，搅拌溶解后，1.2个大气压下加热沸腾20分钟，冷却至室温，加入叠氮钠200mg，过滤，分装保存。
实施例3：
具体方法与实施例1和实施例2相同，所不同的是所用胶体金垫的胶体金直径为20nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为58μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)。将不同浓度的乙酰胆碱酯酶PBST溶液(浓度分别为1ng/ml、10ng/ml、100ng/ml、1μg/ml、10μg/ml)，滴涂到样品垫上，跑样10分钟，肉眼观察，在检测区6和质控区5出现两条明显红线的为阳性结果，只在质控区5出现一条红线着为阴性。
本实施例中，当乙酰胆碱酯酶浓度为1μg/ml时，未出现典型阳性结果(见图6)。
实施例4
具体方法与实施例1和实施例2相同，所不同的是所用胶体金垫的胶体金直径为100nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为45μg/ml；硝酸纤维素膜上检测区6为配对的单克隆抗体(克隆号：1F4-C4-F2-D5)浓度为100μg/ml。将不同浓度的乙酰胆碱酯酶PBST溶液(浓度分别为1ng/ml、10ng/ml、100ng/ml、1μg/ml、10μg/ml)，滴涂到样品垫上，跑样10分钟，肉眼观察，在检测区6和质控区5出现两 条明显红线的为阳性结果，只在质控区5出现一条红线着为阴性。
本实施例中，当乙酰胆碱酯酶浓度为1μg/ml时，未出现典型阳性结果(见图7)。
结论：通过上述的实施例1～4的结果，我们得出，只有当胶体金直径为40nm，与胶体金结合的单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml；单克隆抗体(克隆号：1F4-C4-F2-D5)浓度为100μg/ml时，该检测试纸条在乙酰胆碱酯酶浓度大于等于1μg/ml时，才能检测出典型性阳性结果。因此本发明选取当胶体金直径为40nm，单克隆抗体(克隆号：2H12-F-7)浓度为64μg/ml，单克隆抗体(克隆号：1F4-C4-F2-D5)浓度为100μg/ml。
实验例5
切取一段1mm左右的人脊髓前根，放入250μl的PBS溶液中震荡混匀并离心，获得上清液，然后分别取100μl上清液滴涂到实施例2所获得的胶体金试纸条的样品垫上，跑样10分钟，试纸条上出现典型阳性结果(见图8)；同样取人脊髓后根标本，将其上清液滴涂到实施例2所获得的胶体金试纸条的样品垫上，跑样10分钟，试纸条呈阴性(见图9)。根据以上结果，实施例2所获得的胶体金试纸条可以鉴别出脊髓的前根与后根。根据我们以往免疫传感器与ELISA的实验结果，脊髓前根溶出液的乙酰胆碱酯酶浓度为2.98μg/ml，脊髓后根溶出液的乙酰胆碱酯酶浓度为0.90μg/ml(Sui T1,Ge Y,Liu W,Zhao ZK,Zhang N,Cao X.An acetylcholinesterase antibody-based quartz crystal microbalance for the rapid identification of spinal ventral and dorsal roots.PLoS One.2013Jul 23；8(7):e69049.)。因此，本发明的胶体金试纸条对乙酰胆碱酯酶的检测灵敏度为＞0.90μg/ml。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

  1. 一种用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述诊断试纸条包括设在硬质塑料底板(7)上依次相连的吸水滤纸(1)、检测垫(2)、质控区(5)、检测区(6)、胶体金垫(3)以及样品垫(4)，所述检测垫(2)上的质控区(5)上固定有兔抗鼠IgG，检测垫(2)上的检测区(6)固定有抗乙酰胆碱酯酶单克隆抗体。
  2. 根据权利要求1所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述抗乙酰胆碱酯酶单克隆抗体为小鼠抗乙酰胆碱酯酶抗体。
  3. 根据权利要求1所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述胶体金垫(3)喷涂有胶体金标记的抗乙酰胆碱酯酶单克隆抗体。
  4. 根据权利要求3所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述胶体金标记的抗乙酰胆碱酯酶单克隆抗体的胶体金直径为20nm～100nm、抗乙酰胆碱酯酶单克隆抗体浓度为45μg～100μg/ml。
  5. 根据权利要求3所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述胶体金标记的抗乙酰胆碱酯酶单克隆抗体的胶体金直径为40nm～100nm、抗乙酰胆碱酯酶单克隆抗体浓度为64μg～100μg/ml。
  6. 根据权利要求3所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条，其特征在于，所述胶体金标记的抗乙酰胆碱酯酶单克隆抗体是通过将纯化后的乙酰胆碱酯酶单克隆抗体与大鼠脑组织进行杂交，进而获取特异性神经源性的乙酰胆碱酯酶单克隆抗体作为胶体金标记的备选；然后将获取的单克隆抗体分别进行HRP标记，进行两两组合配对检测，确定可夹心检测标准乙酰胆碱酯酶的抗体对作为配对抗体，即得胶体金标记的抗乙酰胆碱酯酶单克隆抗体。
  7. 权利要求1所述的用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条的制备方法，其特征在于，包括以下步骤：
    1)在硬质塑料底板(7)自上而下依次黏贴吸水滤纸(1)、硝酸纤维素膜的检测垫(2)，胶体金垫(3)和样品垫(4)，真空干燥状态下保存，备用；
    2)按照试纸条组装步骤，分别将抗乙酰胆碱单克隆抗体标记不同直径的胶体金制成胶体金垫，能配对的抗乙酰胆碱单克隆抗体喷点到检测垫的检测区(6)，将兔抗鼠IgG喷点到检测垫的质控区(5)；
    3)将胶体金垫(3)插入样品垫(4)和检测垫(2)之间，将标准乙酰胆碱酯酶的 PBST溶液滴涂到样品垫(4)上，跑样，根据检测区域现象筛选出可用于检测标准乙酰胆碱酯酶的夹心式单克隆抗体组合。
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