Multicolor tracing combined detection method and kit

Abstract

The present invention discloses a multicolor tracing joint detection method and a kit thereof, including preparing a red latex-anti-new coronavirus N protein detection antibody complex, a blue latex-anti-influenza A virus antigen detection antibody complex, and a black latex-anti-influenza B virus antigen detection antibody complex; configuring a conjugate pad and an NC membrane, wherein the conjugate pad contains a colored latex antibody complex coated with a corresponding antibody; using the conjugate pad to combine the antigen in the sample with the colored latex of the corresponding antibody in the conjugate pad to form an antigen-antibody complex, while the antigen-antibody complex is laterally chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane; coloring after enrichment on the NC membrane; judging the type of viral antigen of the current sample according to the color and position of the enriched coloring on the NC membrane. The present invention provides a latex chromatography detection technology for rapid joint diagnosis of multiple antigens, performs joint detection by multicolor tracing, and designs a related kit.

Description

Technical Field

[0001] The invention belongs to a detection method and relates to a multicolor tracing joint detection method and a kit thereof. Background Art

[0002] Tracer technology, after the discovery of radioactive isotopes, tracer method has entered a new stage. Tracer method can show physical processes vividly, intuitively and timely. It can be real tracer or simulated tracer. Tracer method is often used in conjunction with other experimental methods. Brownian motion is a typical example of using tracer method to observe the motion of objects; in particle physics, cloud chambers, bubble chambers and photographic films can all leave traces of particle motion.

[0003] However, there is still much room for improvement in the application of tracer technology in medical testing, especially the current tracer technology, which mostly adopts the form of a single tracking element. The accuracy of lesion judgment in hospital tests needs to be strengthened. The repeated use of different tracer technologies for detection and judgment will increase the patient's pain and has poor timeliness, and cannot form the effect of control detection. Therefore, it is imperative to develop a multi-color tracer joint detection method to increase the speed of medical testing. Summary of the invention

[0004] The present invention solves the problem that the existing tracing technology in the prior art mostly adopts the form of a single tracking element, and the accuracy of judging the lesions in hospital tests needs to be improved, and the repeated use of different tracing technologies for detection and judgment will increase the pain of patients and has poor timeliness, and cannot form the effect of control detection, and provides a multi-color tracing joint detection method.

[0005] The invention also provides a multi-color tracing combined detection kit.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A multi-color tracing joint detection method comprises the following steps:

[0008] 1) Take red latex microspheres, add purified water to dilute and mix, add MES buffer, mix evenly at room temperature, slowly add the detection antibody of the new coronavirus N protein while stirring, start timing after adding, and continue stirring and reacting at room temperature; add EDC solution to the above reaction solution, continue stirring and reacting at room temperature; add latex blocking solution to the above reaction solution, stir and mix at room temperature, immediately homogenize or ultrasonically disperse after adding the latex blocking solution, centrifuge, discard the supernatant, and re-dissolve and resuspend the precipitate with boric acid borax buffer and BSA; homogenize or ultrasonically disperse the resuspended latex, take the dispersed latex, dilute it with purified water, and then detect the particle size to obtain a red latex-antibody complex;

[0009] 2) Prepare the complex of the anti-influenza A virus antigen detection antibody and blue latex microspheres and the complex of the anti-influenza B virus antigen detection antibody and black latex microspheres in the same way as in step 1); then mix these complexes in a ratio of 1:1:1 before use;

[0010] 3) Configure the conjugate pad and the NC membrane. The conjugate pad contains the colored latex antibody complexes coated with the corresponding antibodies obtained in step 1 and step 2. The NC membrane is an NC membrane with a lateral flow chromatography function; dilute the multi-colored latex-labeled detection antibody mixture with a cross-linker diluent, and evenly coat the diluted latex antibody complex mixture on the conjugate pad. The coating amount per glass fiber is 10-50 mL, and dry it at 45 °C for 24 h;

[0011] 4) Use the conjugate pad to combine the antigen in the sample with the colored latex of the corresponding antibody in the conjugate pad to form an antigen-antibody complex. At the same time, the antigen-antibody complex laterally chromatographs on the NC membrane and forms a double antibody sandwich complex with the capture antibody coated on the NC membrane;

[0012] 5) After the double antibody sandwich complex is enriched on the NC membrane, it develops color;

[0013] 6) Judge the type of virus antigen in the current sample according to the color and position of the enriched and developed color on the NC membrane.

[0014] As a preferred embodiment of the present invention, in step 1), the particle size of the red latex microspheres is 100 nm - 500 nm; in step 2), the particle size of the blue latex microspheres is 100 nm - 500 nm; the particle size of the black latex microspheres is 100 nm - 500 nm.

[0015] As a preferred embodiment of the present invention, in step 1), the addition amount of the MES buffer solution is 1.0 - 5.0 mL, the concentration is 100 - 500 mM, and the pH is 4.5 - 6.5; the concentration of the EDC solution is 10 mg / mL, and the addition amount is 0.1 - 0.15 mL;

[0016] As a preferred embodiment of the present invention, in step 1), the latex blocking solution is prepared from 1 - 3 g of BSA, 0.1 mL of Proclin-300 and purified water, and the addition amount is 0.1 - 0.5 mL.

[0017] As a preferred embodiment of the present invention, in step 1), the parameters of homogenization are 600 - 1500 Bar; the parameters of ultrasonic treatment are 100 - 200 w, 1 - 2 s * 10 - 20 times, with an interval of 4 s.

[0018] As a preferred embodiment of the present invention, in step 1), the conditions for qualified particle size detection are that the particle size is less than 500 nm and the PDI is less than 0.1.

[0019] As a preferred embodiment of the present invention, in step 1), the centrifugation parameters are 10-25°C, 23000g for 30 min, and the precipitate is redissolved and resuspended using 5-15 mL of 0.05-0.2 M boric acid borax buffer (pH 7.0-9.0) and 1.0-5.0 mL of 50-200 mg / mL BSA.

[0020] As a preferred embodiment of the present invention, in step 3), the mass ratio of the red latex-antibody complex, the blue latex-antibody complex and the black latex-antibody complex is 1:1:1.

[0021] The present invention also provides a multicolor tracing joint detection kit, comprising a binding pad for binding to a sample, an NC membrane, a back plate and a water absorbent layer, wherein the middle part of the back plate is provided with the NC membrane, one side of the NC membrane is overlapped with the binding pad, the outer side of the binding pad is provided with a water absorbent pad, and the other side of the NC membrane is also provided with a water absorbent pad, the binding pad contains several kinds of the above-mentioned colored latex antibody complexes coated with corresponding antibodies, the antigen in the sample is combined with the colored latex of the corresponding antibody in the binding pad to form an antigen-antibody complex, and at the same time, it is laterally chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane, and the type of viral antigen in the current sample is determined by color development after enrichment on the NC membrane.

[0022] As a preferred embodiment of the present invention, the conjugate pad contains three colored latexes coated with corresponding antibodies, namely, red latex-anti-new coronavirus N protein detection antibody complex, blue latex-anti-influenza A virus antigen detection antibody complex, and black latex-anti-influenza B virus antigen detection antibody complex.

[0023] The substantial effect of the present invention is as follows: the present invention relates to the field of immunodiagnosis, and in view of the problem of single antigen detection items and low efficiency, provides a latex chromatography detection technology for rapid joint diagnosis of multiple antigens, performs joint detection through multi-color tracing, and designs related kits. Clinical application and evaluation of the multiple antigen joint rapid detection method of latex immunochromatography: collect qualified clinical specimens, compare the established new method with the existing gold standard method, statistically analyze the results, and combine clinical indications, imaging, etc. to prove that the present invention can detect antigens quickly and sensitively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a schematic flow chart of a method.

[0025] Figure 2 The figure is a schematic diagram of the structure of the multi-color tracing combined detection kit of the present invention.

[0026] Figure 3These are the results of different viruses tested by the three antigen detection kits of the present invention.

[0027] Figure 4 It is the color intensity of inactivated viruses of different concentrations tested by the three antigen detection kits of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below through specific embodiments.

[0029] Embodiment 1:

[0030] See also Figure 1 This embodiment provides a multi-color tracing joint detection method, including the following method:

[0031] Step 1: Take 1.0 mL of red latex microspheres with a particle size of 300 nm-380 nm, add them to 14.0 mL of purified water to dilute and mix, add 5.0 mL of 100 mM PH6.1 MES buffer, mix evenly at room temperature, then slowly add an appropriate amount of detection antibody for the new coronavirus N protein while stirring, start timing after addition, and continue stirring and reacting at room temperature for 15 minutes; add 0.1375 mL of 10 mg / mL EDC solution to the above reaction solution, and continue stirring and reacting at room temperature for 60 minutes; add 0.404 mL of latex blocking solution (ingredients: 3 g BSA + 0.1 mL Proclin-300 + 99.9 mL purified water) to the above reaction solution, stir and mix at room temperature for 60 minutes, and immediately disperse by homogenization (600-1500 Bar) or ultrasound (200 w, 2s*10 times, 4s interval) after adding the latex blocking solution. Centrifuge at 23000g for 30min at 10-25°C, discard the supernatant carefully, and use 12.0mL of 0.2M boric acid borax buffer and 3.0mL of 200mg / mL BSA to redissolve and resuspend the precipitate; disperse the above latex by homogenization (600-1500Bar) or ultrasound (200w, 2s*10 times, 4s interval); take 1uL of the dispersed latex, dilute it with 1.0mL of purified water, and then detect the particle size. The particle size is less than 380nm and the PDI is less than 0.1, which is qualified; the red latex-antibody 1 complex can be stored in the dark at 2-8°C for 3 months.

[0032] Step 2: Using the same method, a complex of an anti-influenza A virus antigen detection antibody and blue latex microspheres (particle size of 300nm-380nm) and a complex of an anti-influenza B virus antigen detection antibody and black latex microspheres (particle size of 300nm-380nm) are prepared.

[0033] Then mix these compounds in a 1:1:1 ratio before use.

[0034] Step three, prepare a conjugate pad and an NC membrane, wherein the conjugate pad contains several kinds of colored latex coated with corresponding antibodies, and the NC membrane is an NC membrane with lateral chromatography function; dilute the multicolor latex-labeled detection antibody mixture with a cross-linking diluent, the dilution ratio is 1:12.5, and the diluted latex-antibody complex mixture is evenly coated on the conjugate pad, the coating amount for each glass fiber is 30 mL, and dry at 45°C for 24 hours.

[0035] Step 4: The antigen in the sample is combined with the colored latex of the corresponding antibody in the binding pad to form an antigen-antibody complex. At the same time, the antigen-antibody complex is lateral chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane;

[0036] Step 5, the double antibody sandwich complex is enriched on the NC membrane and then developed;

[0037] Step six, determine the type of viral antigen in the current sample based on the color and position of the enriched color on the NC membrane.

[0038] Example 2

[0039] See also Figure 2 This embodiment provides a multicolor tracing joint detection kit, including a conjugate pad 2 for binding with a sample, an NC membrane 3, a back plate 1 and a water absorbent pad 4, wherein the middle part of the back plate 1 is provided with an NC membrane 3, one side of the NC membrane 3 is overlapped with the conjugate pad 2, the outer side of the conjugate pad 2 is provided with a sample pad 5, and the other side of the NC membrane 3 is also provided with a water absorbent pad, wherein the conjugate pad contains several kinds of color latex coated with corresponding antibodies, and the antigen in the sample is combined with the color latex of the corresponding antibody in the conjugate pad to form an antigen-antibody complex, and at the same time, the antigen is lateral chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane, and the type of viral antigen in the current sample is determined by color development after enrichment on the NC membrane. The conjugate pad contains three kinds of color latex coated with corresponding antibodies, namely, a red latex-anti-new coronavirus N protein detection antibody complex 6, a blue latex-anti-influenza A virus antigen detection antibody complex 7, and a black latex-anti-influenza B virus antigen detection antibody complex 8.

[0040] See also Figure 3 , In the figure, A: ℃ OVID-19 antigen positive; B: influenza A antigen positive; ℃: influenza B antigen positive; D: negative; E: invalid test.

[0041] See also Figure 4 AF plates show the color intensity of different concentrations of inactivated viruses tested by the three antigen detection kits produced by the present invention (A: control; B: 1.0×10 6 TCID50 / mL℃OVID-19+1.0×104 TCID50 / mL influenza A+1.0×10 4 TCID50 / mL; C: 1.0×10 5 TCID50 / mL℃OVID-19+5.0×10 3 TCID50 / mL influenza A+5.0×10 3 TCID50 / mL; D: 1.0×10 4 TCID50 / mL℃OVID-19+1.0×10 3 TCID50 / mL influenza A+1.0×10 3 TCID50 / mL; E: 1.0×10 3 TCID50 / mL℃OVID-19+5.0×10 2 TCID50 / mL influenza A+5.0×10 2 TCID50 / mL; F: 1.0×10 2 TCID50 / mL℃OVID-19+1.0×10 2 TCID50 / mL influenza A+1.0×10 2 TCID50 / mL.

[0042] This embodiment relates to the field of immunodiagnosis. Aiming at the problem of single antigen detection items and low efficiency, a latex chromatography detection technology for rapid joint diagnosis of multiple antigens is provided, and joint detection is performed through multi-color tracing, and related kits are designed. Clinical application and evaluation of the multiple antigen joint rapid detection method of latex immunochromatography: Collect qualified clinical specimens, compare the established new method with the existing gold standard method, statistically analyze the results, and combine clinical indications, imaging, etc. to prove that this method can detect antigens quickly and sensitively.

[0043] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A multi-color tracing joint detection method, It is characterized in that The following steps are involved: 1) Take red latex microspheres, add purified water to dilute and mix, add MES buffer, mix evenly at room temperature, slowly add the detection antibody of the new coronavirus N protein while stirring, start the time after adding, and continue stirring the reaction at room temperature; add EDC solution to the reaction solution, continue stirring the reaction at room temperature; add latex blocking solution to the above reaction solution, stir and mix at room temperature, immediately homogenize or ultrasonically disperse after adding the latex blocking solution, centrifuge, discard the supernatant, and re-dissolve and re-suspend the precipitate with boric acid borax buffer and BSA; homogenize or ultrasonically disperse the resuspended latex, take the dispersed latex, dilute it with purified water, and then detect the particle size to obtain a red latex-antibody complex; 2) preparing a complex of an anti-influenza A virus antigen detection antibody and blue latex microspheres and a complex of an anti-influenza B virus antigen detection antibody and black latex microspheres by the same method as step 1); and then mixing these complexes in a ratio of 1:1:1 before use; 3) preparing a conjugate pad and a NC membrane, wherein the conjugate pad contains the colored latex antibody complex coated with the corresponding antibody obtained in step 1) and step 2), and the NC membrane is an NC membrane with lateral flow chromatography function; diluting the multicolor latex-labeled detection antibody mixture with a cross-linking diluent, and evenly coating the diluted latex antibody complex mixture on the conjugate pad, with a coating amount of 30 mL per glass fiber, and drying at 45° C. for 24 hours; 4) The antigen in the sample is combined with the colored latex of the corresponding antibody in the binding pad to form an antigen-antibody complex. At the same time, the antigen-antibody complex is lateral chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane; 5) The double antibody sandwich complex is enriched on the NC membrane and then colored; 6) Determine the type of viral antigen in the current sample based on the color and position of the enriched color on the NC membrane.

2. A multi-color tracing joint detection method according to claim 1, It is characterized in that In step 1), the particle size of the red latex microspheres is 300nm-380nm; in step 2), the particle size of the blue latex microspheres is 300nm-380nm; and the particle size of the black latex microspheres is 300nm-380nm.

3. A multicolor tracing joint detection method according to claim 1, It is characterized in that In step 1), the amount of MES buffer added was 5.0 mL, the concentration was 100 mM, and the pH was 6.1; the concentration of EDC solution was 10 mg / mL, and the amount added was 0.1375 mL.

4. A multi-color tracing joint detection method according to claim 1, It is characterized in that In step 1), the latex blocking solution is prepared from 3 g BSA, 0.1 mL Proclin-300 and 99.9 mL purified water, and the added amount is 0.404 mL.

5. A multi-color tracing joint detection method according to claim 1, It is characterized in that In step 1), the homogenization parameters are 600-1500 Bar; the ultrasonic parameters are 200w, 2s*10 times, and the interval is 4s.

6. A multi-color tracing joint detection method according to claim 1, It is characterized in that In step 1), the conditions for qualified particle size detection are that the particle size is less than 380nm and the PDI is less than 0.

1.

7. A multi-color tracing joint detection method according to claim 1, It is characterized in that In step 3), the mass ratio of the red latex-antibody complex, the blue latex-antibody complex and the black latex-antibody complex is 1:1:

1.

8. A multi-color tracing combined detection kit, It is characterized in that The invention comprises a binding pad for binding with a sample, an NC membrane, a back plate and a water-absorbing layer, wherein the middle part of the back plate is provided with the NC membrane, one side of the NC membrane is overlapped with the binding pad, the outer side of the binding pad is provided with a water-absorbing pad, and the other side of the NC membrane is also provided with a water-absorbing pad, the binding pad contains several kinds of color latex antibody complexes coated with corresponding antibodies as claimed in any one of claims 1 to 7, the antigen in the sample is combined with the color latex of the corresponding antibody in the binding pad to form an antigen-antibody complex, and at the same time, the antigen is laterally chromatographed on the NC membrane to form a double antibody sandwich complex with the capture antibody coated on the NC membrane, and the type of the virus antigen in the current sample is determined by color development after enrichment on the NC membrane.

9. A multicolor tracing combined detection kit according to claim 8, It is characterized in that The conjugate pad contains three colored latexes coated with corresponding antibodies, namely, red latex-anti-new coronavirus N protein detection antibody complex, blue latex-anti-influenza A virus antigen detection antibody complex, and black latex-anti-influenza B virus antigen detection antibody complex.

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