Single-molecule immunodetection raw material and detection method thereof

Abstract

The invention belongs to the field of single-molecule immunodetection, and discloses a single-molecule immunodetection raw material which comprises a detection antibody and a capture antibody, the detection antibody and the capture antibody are coupled with an azide functional group in a fixed-point manner, and the detection antibody and the capture antibody coupled with a sodium azide functional group are respectively coupled with an oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), an oligonucleotide sequence 2 modified with dibenzocyclooctyne (DBCO) and an oligonucleotide sequence 3 modified with dibenzocyclooctyne (DBCO). The sequence is as shown in SEQ ID NO.1 and is coupled with the oligonucleotide sequence 2, and the sequence is as shown in SEQ ID NO.2. The invention also discloses a single-molecule immunodetection method. According to the single-molecule immunodetection raw material and the single-molecule immunodetection method, the detection of the femtogram-level biomarker can be realized. The whole detection process can be completed under the condition of normal temperature in a laboratory, variable-temperature incubation is not needed, special equipment is not needed, and results can be interpreted by being matched with a conventional flow cytometer or a fluorescence microscope. Customized chips and consumables are not needed, and the cost is controllable. A double-antibody sandwich method is applied, so that the detection specificity is higher.

Description

Technical Field

[0001] The invention belongs to the field of single molecule immunoassay, and specifically relates to a single molecule immunoassay raw material and a detection method thereof. Background Art

[0002] Early diagnosis of diseases, accurate diagnosis, and reduction of severe disease rates are feasible methods to alleviate the medical burden and improve the quality of human life. Immunodiagnosis is the application of immunological theories, techniques, and methods to diagnose various diseases and measure immune status. There are several major categories of immunodiagnostic technologies: radioimmunoassay, enzyme-linked immunosorbent assay, colloidal gold, fluorescent immunoassay, time-resolved fluorescence, and chemiluminescence. Early diagnosis, early detection, and early treatment are the main means to improve the treatment effects of many diseases. Commonly used immunological detection methods in clinical practice, such as enzyme-linked immunosorbent assay and chemiluminescence assay, have a detection sensitivity limited to 10 -14 ~10 -12 nmol / L, which cannot meet the needs of early diagnosis. Single-molecule immunity is a new type of immunoassay method based on the theoretical basis of Poisson distribution. The sensitivity of detection can reach the single-molecule level, which can be applied to the early diagnosis, medication guidance and later disease monitoring of various diseases. The single-molecule immunoassay system based on β-galactosidase-catalyzed signal amplification represented by Quanterix in the world counts positive signals by loading a magnetic bead in a micropore. In the commercialized Simoa HD-1 analyzer, the magnetic beads are loaded and isolated by gravity, but only 5% of the magnetic beads are separated and analyzed. Therefore, it also leads to large repetition differences, poor reagent stability, high cost and other problems, which is not suitable for large-scale productization and application promotion. Suzhou Yuce Biotechnology Co., Ltd. in China uses in situ signal enhancement nanoparticles as signal amplification and has developed corresponding single-molecule immunoassay instruments. However, the resistance to linking the in situ signal-enhancing nanoparticles and magnetic beads through a single protein molecule is large, the stability of the detection system is poor, the coefficient of variation of the detection results is large, and it is difficult to distinguish the number of in situ signal-enhancing nanoparticles bound to the magnetic beads, which narrows the detection range of single-molecule immunity. The above two single-molecule detection technologies require dedicated detection and analysis equipment, which has high equipment R&D costs and is difficult to promote clinically.

[0003] The existing single-molecule detection technology has many problems, such as extremely high equipment precision requirements, difficulty in developing detection reagents, poor stability, high precision of auxiliary consumables and chips, difficult to control costs, poor repeatability of detection results, etc. These problems seriously hinder the application of single-molecule detection technology in scientific research and medical diagnosis markets. Summary of the invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention provides a single molecule immunoassay raw material, including a detection antibody and a capture antibody, wherein the detection antibody and the capture antibody are site-specifically coupled to an azide functional group, and the detection antibody and the capture antibody coupled to a sodium azide functional group are respectively coupled to an oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), the sequence of which is shown in SEQ ID NO.1, and an oligonucleotide sequence 2, the sequence of which is shown in SEQ ID NO.2.

[0005] Furthermore, the capture antibody coupled with the oligonucleotide sequence is coated on the magnetic beads.

[0006] The present invention also provides a single molecule immunoassay method, comprising the following steps:

[0007] Step 1, raw material preparation: preparing the single molecule immunoassay raw material, including:

[0008] Step 1.1 site-specific coupling of antibody and oligonucleotide;

[0009] Step 1.2: Coating magnetic beads with capture antibodies coupled with oligonucleotide sequences;

[0010] Step 2, configuring a single molecule immune response system;

[0011] Step 3: Connect the reaction system to form a rolling circle amplification template

[0012] Step 4: Rolling circle amplification for detection signal amplification

[0013] Step 5: Count the fluorescence signals.

[0014] Furthermore, step 1.1 is specifically as follows: according to the detection marker, a suitable antibody pair is selected, which is divided into a detection antibody and a capture antibody; the detection antibody and the capture antibody are site-specifically coupled to an azide functional group by a one-step enzyme catalysis method; the detection antibody and the capture antibody coupled with a sodium azide functional group are respectively coupled with oligonucleotide sequence 1 and oligonucleotide sequence 2 modified with dibenzocyclooctyne (DBCO), and the coupling method is based on the principle of click chemistry reaction; free oligonucleotides are removed by ultrafiltration, and the concentration of the prepared antibody-oligonucleotide complex is quantitatively detected by BCA.

[0015] Furthermore, step 1.2 is as follows: select 2-10 μm magnetic beads with fluorescently labeled amino functional groups; couple the capture antibody coupled with the oligonucleotide sequence to the above magnetic beads through a reverse click chemistry reaction; and remove the capture antibody not coated on the magnetic beads with a magnetic bead washing buffer.

[0016] Furthermore, step 2 is as follows:

[0017] The magnetic beads coated with the capture antibody were incubated with the sample at room temperature for 1 hour, the liquid was removed, and the sample was washed with a washing buffer; the washing buffer was removed, and the detection antibody coupled with oligonucleotide 1 was added and incubated with the capture antibody-coated magnetic beads, and the sample was incubated at room temperature for 1 hour; the liquid was removed, and the sample was washed with a washing buffer.

[0018] Furthermore, step 3 is as follows:

[0019] Prepare a ligation reaction system, including a linker, a ligase, and a ligase working buffer, and incubate with the magnetic beads in step 2 at room temperature for 30 minutes; remove the incubation reaction solution and add a washing buffer for washing.

[0020] Further, step 4, rolling circle amplification for detection signal amplification

[0021] Prepare a rolling circle amplification reaction system, including isothermal amplification enzyme, amplification substrate dNTP, amplification reaction buffer Mg2+ and a fluorescent signal probe hybridized with the rolling circle amplification template; incubate at room temperature for 30 minutes to 60 minutes; remove the incubation reaction solution, add washing buffer for washing, remove the washing buffer, and resuspend the magnetic beads in step 3 with magnetic bead resuspension buffer.

[0022] Furthermore, step 5 is as follows:

[0023] The magnetic beads obtained in step 4 were dropped onto a 1 cm*1 cm area of ​​the glass slide, and the entire area where the magnetic beads were dropped was scanned under a fluorescence scanning microscope to count the positive signal points.

[0024] The present invention can detect femtogram-level biomarkers through single-molecule immunoassay raw materials and detection methods. The method can complete the entire detection process under normal laboratory temperature conditions, without the need for variable temperature incubation or special equipment, and can be matched with a conventional flow cytometer or fluorescence microscope to interpret the results. No customized chips and consumables are required, and the cost is controllable. The double antibody sandwich method is used to achieve higher detection specificity. The rolling circle amplification signal amplification technology is integrated to improve the accuracy of single molecule signal recognition.

[0025] The detection method of the present invention combines the non-site coupling of the capture antibody with the solid phase magnetic beads, the capture antibody and the detection antibody are site-coupled with the specific sequence oligonucleotide sequence by a one-step enzyme catalysis method, and the detection signal is amplified by rolling circle amplification to form an efficient detection system, which can be used for the detection of protein markers in samples such as serum, plasma, tissue fluid, and cerebrospinal fluid. Compared with the current single molecule immunoassay method, this method has the advantages of high sensitivity, strong specificity, and a simpler and more intuitive detection method, which improves the stability of the detection system and the repeatability of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the process of the single molecule immunoassay method of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] like Figure 1 As shown, the single molecule immunoassay method of the present invention comprises the following steps:

[0029] Step 1, raw material preparation:

[0030] Step 1.1 Site-directed conjugation of antibodies and oligonucleotides

[0031] According to the detection marker, a suitable antibody pair is selected, which is divided into a detection antibody and a capture antibody; the detection antibody and the capture antibody are site-specifically coupled to an azide functional group by a one-step enzyme catalysis method; the detection antibody and the capture antibody coupled with a sodium azide functional group are respectively coupled with an oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), the sequence is shown in SEQ ID NO.1 and an oligonucleotide sequence 2, the sequence is shown in SEQ ID NO.2, and the coupling method is based on the principle of click chemistry reaction; free oligonucleotides are removed by ultrafiltration, and the concentration of the prepared antibody-oligonucleotide complex is quantitatively detected by BCA.

[0032] Step 1.2 Coating magnetic beads with capture antibody coupled with oligonucleotide sequence

[0033] Select 2-10 μm magnetic beads with fluorescently labeled amino functional groups; couple the capture antibody coupled with the oligonucleotide sequence to the above magnetic beads through a reverse click chemistry reaction; and remove the capture antibody not coated on the magnetic beads by using magnetic bead washing buffer 1.

[0034] Step 2: Configure the single molecule immune response system

[0035] The magnetic beads coated with the capture antibody are incubated with the sample at room temperature for 1 hour, the liquid is removed, and the sample is washed with washing buffer 2; the washing buffer 2 is removed, and the detection antibody coupled with oligonucleotide 1 is added and incubated with the capture antibody-coated magnetic beads, and the mixture is incubated at room temperature for 1 hour; the liquid is removed, and the sample is washed with washing buffer 2.

[0036] Step 3: Connect the reaction system to form a rolling circle amplification template

[0037] Prepare a ligation reaction system, including a linker, a ligase, a ligase working buffer, etc., and incubate with the magnetic beads in step 2 at room temperature for 30 minutes; remove the incubation reaction solution, and add washing buffer 3 for washing.

[0038] Step 4: Rolling circle amplification for detection signal amplification

[0039] A rolling circle amplification reaction system is prepared, including an isothermal amplification enzyme, an amplification substrate dNTP, an amplification reaction buffer Mg2+, etc., and a fluorescent signal probe hybridized with the rolling circle amplification template; incubate at room temperature for 30 minutes to 60 minutes; remove the incubation reaction solution, add washing buffer 4 for washing, remove the washing buffer 4, and resuspend the magnetic beads in step 3 with a magnetic bead resuspension buffer.

[0040] Step 5: Fluorescence signal counting

[0041] The magnetic beads obtained in step 4 were dropped onto a 1 cm*1 cm area of ​​the glass slide, and the entire area where the magnetic beads were dropped was scanned under a fluorescence scanning microscope to count the positive signal points.

[0042] Example

[0043] Taking the detection of IL-6 in plasma samples as an example, the operation process of the single molecule immunoassay method of the present invention is as follows:

[0044] 1. The IL-6 detection antibody and the capture antibody are replaced by the principle of buffer exchange in the dialysis bag. The original antibody storage solution is replaced with 25mM Tris-HCL buffer with a pH value of 7.2, and the antibody concentration is adjusted to 1-10mg / ml with Tris-HCL buffer.

[0045] 2. Mix the total amount of 1 mg of the above IL-6 detection antibody with Endo-S enzyme at a mass ratio of 1:5 to 1:20, and then mix the substrate disaccharide oxazoline (N3-LacNAc-oxa) with the antibody at a molar ratio of 10:1 to 50:1, incubate at room temperature for 30 to 60 minutes, and the proportion of antibody sugar chain catalytic binding azide groups can reach more than 90%. The above IL-6 capture antibody catalyzes azide groups on sugar chains in the same way.

[0046] 3. The above-mentioned catalytic azide group detection antibody reaction system is subjected to buffer replacement by a desalting column or a dialysis bag, and the above-mentioned catalytic buffer is replaced with a phosphate buffer, the pH is kept neutral, and the molar concentration of phosphate is 10mM. The above-mentioned catalytic azide group capture antibody is treated in the same manner.

[0047] 4. Synthesize oligonucleotides 1 and 2 with dibenzocyclooctyne (DBCO) modification at the end, the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the oligonucleotides are dissolved in ultrapure water or phosphate buffer to 100-200 micromoles. The IL-6 detection antibody prepared in the above step is incubated with oligonucleotide sequence 1 at a molar ratio of 1:2 to 1:10 at room temperature for 1-2 hours, or at 4 degrees Celsius overnight; similarly, the IL-6 capture antibody prepared in the above step is incubated with oligonucleotide sequence 2.

[0048] 5. The detection antibody reaction system incubated with oligonucleotide sequence 1 is subjected to buffer replacement through an ultrafiltration tube or a dialysis bag to remove excess free oligonucleotide sequence 1, and the coupling buffer is replaced with a phosphate buffer, the pH is kept neutral, and the phosphate molar concentration is 10mM. The capture antibody reaction system incubated with oligonucleotide sequence 2 in the above step is treated in the same manner.

[0049] 6. Select 2-10 μm magnetic microspheres with fluorescently labeled amino functional groups, wash the magnetic microspheres three times with washing buffer 1, 3 minutes each time, remove the washing buffer with a magnetic rack, and re-wash the magnetic microspheres with neutral 10 mM phosphate solution.

[0050] 7. The tetrazine-NHS compound reacts with the amino groups on the magnetic beads. The molecular ratio of the compound to the magnetic beads is 1 million to 2 million:1. Incubate with rotation at room temperature for 2 hours. Wash with wash buffer 2 three times for 3 minutes each time. Remove the wash buffer 2 with a magnetic rack and re-incubate the magnetic beads with a neutral 10 mM phosphate solution.

[0051] 8. The trans-octene-NHS compound is reacted with the capture antibody coupled to the oligonucleotide sequence 2 obtained in the above step 5, and the molar ratio of the trans-octene-NHS compound to the antibody is 1:5 to 1:50. The mixture is incubated at room temperature for 2 hours, and the buffer is replaced by a neutral 10 mM phosphate solution through a dialysis bag or a desalting column.

[0052] 9. Incubate the magnetic beads in step 7 above with the capture antibody in step 8 at a ratio of 1:100,000 to 1,000,000 at room temperature for 2 hours, or overnight at 4 degrees Celsius. Wash three times with wash buffer 1 for 3 minutes each to remove excess antibody. Rewash the magnetic beads with wash buffer 2.

[0053] 10. Incubate the magnetic beads in step 9 with the sample, with a sample volume of 10-100ul and a number of 100,000-1,000,000 magnetic beads. Incubate at room temperature for 1 hour with rotation, remove the incubation liquid, and wash 5 times with washing buffer 2, 3 minutes each time. Resuspend with washing buffer 2.

[0054] 11. Incubate the magnetic beads in step 10 above with the detection antibody prepared in step 4, with the ratio of antibody to magnetic beads being 100-100,000:1. Incubate at room temperature for 1 hour with rotation, remove the incubation liquid, and wash 5 times with washing buffer 2, 3 minutes each time.

[0055] 12. Prepare a ligation reaction system, with 50-200 pM of both connector 1 and connector 2, 0.05 to 0.5 U / μl of T4 DNA ligase, the sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, 150 mM NaCl, 0.1% BSA, 0.05% Tween-20, 10 mM Mg2+, 10 mM Tris-HCL, PH7.2. Resuspend the magnetic beads in step 11 above in the prepared ligation reaction system. Incubate at room temperature for 30 minutes. Remove the incubation reaction solution and wash 5 times with washing buffer 3, 3 minutes each time.

[0056] 13. Prepare a rolling circle amplification reaction system, phi29 DNA polymerase 0.1-0.5U / μL, 10mM MgCl2, 10mM (NH4)2SO4, 200μM dNTPs, 0.1% BSA, 0.05% Tween-20, 1-10nM fluorescent detection probe, as shown in SEQ ID NO.5, 250mM Tris-HCL, PH7.2. Resuspend the magnetic beads in the above step 12 in the rolling circle amplification reaction system, and incubate at room temperature for 30 to 60 minutes with rotation. Remove the incubation reaction solution by magnetic rack, and wash 5 times with washing buffer 4, 3 minutes each time.

[0057] 14. Use an immunohistochemistry pen to circle a 1*1 cm area on the slide in advance, resuspend the magnetic beads obtained in step 13 above with resuspension buffer and drop them into the area, and scan the target area with a fluorescent scanner.

[0058]

[0059] Table 1. Buffer composition.

Claims

1. A single molecule immunoassay raw material, comprising a detection antibody and a capture antibody, characterized in that: The detection antibody and the capture antibody are site-specifically coupled to azide functional groups, and the detection antibody and the capture antibody coupled to sodium azide functional groups are respectively coupled to oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), the sequence of which is shown in SEQ ID NO.1, and oligonucleotide sequence 2, the sequence of which is shown in SEQ ID NO.

2.

2. The single molecule immunoassay raw material according to claim 1, characterized in that: Capture antibodies conjugated with oligonucleotide sequences are coated on magnetic beads.

3. A single molecule immunoassay method comprising the following steps: Step 1, raw material preparation: preparing the single molecule immunoassay raw material as claimed in claim 1 or 2, comprising: Step 1.1 site-specific coupling of antibody and oligonucleotide; Step 1.2: Coating magnetic beads with capture antibodies coupled with oligonucleotide sequences; Step 2, configuring a single molecule immune response system; Step 3: Connect the reaction system to form a rolling circle amplification template Step 4: Rolling circle amplification for detection signal amplification Step 5: Count the fluorescence signals.

4. The single molecule immunoassay method according to claim 3, characterized in that: Step 1.1 is as follows: according to the detection marker, a suitable antibody pair is selected, which is divided into a detection antibody and a capture antibody; the detection antibody and the capture antibody are site-specifically coupled to an azide functional group by a one-step enzyme catalysis method; the detection antibody and the capture antibody coupled with a sodium azide functional group are coupled with oligonucleotide sequence 1 and oligonucleotide sequence 2 modified with dibenzocyclooctyne (DBCO), respectively, and the coupling method is based on the principle of click chemistry reaction; free oligonucleotides are removed by ultrafiltration, and the concentration of the prepared antibody-oligonucleotide complex is quantitatively detected by BCA.

5. The single molecule immunoassay method according to claim 3, characterized in that: Step 1.2 is as follows: select 2-10 μm magnetic beads with fluorescently labeled amino functional groups; couple the capture antibody coupled with the oligonucleotide sequence to the above magnetic beads through a reverse click chemistry reaction; and remove the capture antibody not coated on the magnetic beads with a magnetic bead washing buffer.

6. The single molecule immunoassay method according to claim 3, characterized in that: Step 2 is as follows: The magnetic beads coated with the capture antibody were incubated with the sample at room temperature for 1 hour, the liquid was removed, and the sample was washed with a washing buffer; the washing buffer was removed, and the detection antibody coupled with oligonucleotide 1 was added and incubated with the magnetic beads coated with the capture antibody, and the incubation was continued at room temperature for 1 hour; Remove liquid and wash with wash buffer.

7. The single molecule immunoassay method according to claim 3, characterized in that: Step 3 is as follows: Prepare a ligation reaction system, including a linker, a ligase, and a ligase working buffer, and incubate with the magnetic beads in step 2 at room temperature for 30 minutes; remove the incubation reaction solution and add a washing buffer for washing.

8. The single molecule immunoassay method according to claim 3, characterized in that: Step 4 is as follows: Prepare a rolling circle amplification reaction system, including isothermal amplification enzyme, amplification substrate dNTP, amplification reaction buffer Mg2+ and a fluorescent signal probe hybridized with the rolling circle amplification template; incubate at room temperature for 30 minutes to 60 minutes; remove the incubation reaction solution, add washing buffer for washing, remove the washing buffer, and resuspend the magnetic beads in step 3 with magnetic bead resuspension buffer.

9. The single molecule immunoassay method according to claim 3, characterized in that: Step 5 is as follows: The magnetic beads obtained in step 4 were dropped onto a 1 cm*1 cm area of ​​the glass slide, and the entire area where the magnetic beads were dropped was scanned under a fluorescence scanning microscope to count the positive signal points.

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