A single molecule immunoassay material and a method for detecting the same

Abstract

The application belongs to the field of single molecule immunodetection, and discloses a single molecule immunodetection raw material, which comprises detection antibodies and capture antibodies, wherein the detection antibodies and the capture antibodies are coupled with azide functional groups, the detection antibodies and the capture antibodies coupled with sodium azide functional groups are respectively coupled with oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), and the sequence is shown as SEQ ID NO. 1, and oligonucleotide sequence 2, and the sequence is shown as SEQ ID NO. 2. The application further discloses a single molecule immunodetection method. The application can realize the detection of a few grams of biomarkers through the single molecule immunodetection raw material and the detection method. The entire detection process can be completed under the normal temperature condition in a laboratory, without the need of temperature incubation and special equipment, and the result can be read by matching a conventional flow cytometer or a fluorescence microscope. The application does not need to customize chips and consumables, and the cost is controllable. The application uses a double antibody sandwich method, and the specificity is higher.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of single molecule immunoassay, and particularly relates to a single molecule immunoassay raw material and a detection method thereof. BACKGROUND

[0002] Early diagnosis, accurate diagnosis, and reduction of severe illness rate are feasible methods for relieving medical burden and improving human living quality. Immune diagnosis is a method for diagnosing various diseases and measuring immune state by applying the theory, technology and method of immunology. There are several categories of immune diagnosis technologies: radioimmunoassay, enzyme-linked immunoassay, colloidal gold, fluorescent immunoassay, time-resolved fluorescence, and chemiluminescence. Early diagnosis, early detection, and early treatment are main means for improving the treatment effect of various diseases. The detection sensitivity of commonly used clinical immunological detection methods, such as enzyme-linked immunosorbent assay and chemiluminescence method, is limited to 10 -14 ~ 10 -12 nmol / L, which cannot meet the needs of early diagnosis. Single molecule immunoassay is a new type of immunoassay method based on Poisson distribution, and the detection sensitivity can reach the single molecule level, which can be applied to early diagnosis, medication guidance and later disease monitoring of various diseases. The single molecule immunoassay system based on beta-galactosidase catalytic signal amplification represented by Quanterix Company in the international market is a method for counting positive signals by loading a magnetic bead in a microwell. 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, which also leads to large differences in repeatability, poor stability of reagents, high cost and other problems, and is not suitable for large-scale productization and application promotion. Suzhou Yumei Biotechnology Co., Ltd. uses in-situ signal enhancement nanoparticles as signal amplification and develops a corresponding single molecule immunoassay instrument. However, the resistance to be overcome by connecting the in-situ signal enhancement nanoparticles and the magnetic beads through a single protein molecule is large, the stability of the detection system is poor, the coefficient of variation of the detection result is large, and it is difficult to distinguish the number of in-situ signal enhancement nanoparticles combined with the magnetic beads, which makes the detection range of single molecule immunoassay narrow. The above two kinds of single molecule detection technologies need special detection and analysis equipment, and the equipment development cost is high, which makes it difficult to promote clinically.

[0003] The existing single molecule detection technology has problems such as high precision requirement of equipment, great difficulty in development of detection reagent, poor stability, high precision of auxiliary consumables and chip, difficult cost control, and poor repeatability of detection results. These problems seriously hinder the application of single molecule detection technology in the market of scientific research and medical diagnosis. SUMMARY

[0004] In order to solve the above technical problems in the prior art, the present application provides a single molecule immunodetection raw material, comprising detection antibodies and capture antibodies, the detection antibodies and the capture antibodies are coupled with azide functional groups, the detection antibodies and the capture antibodies coupled with sodium azide functional groups are respectively coupled with oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), and the sequence is shown as SEQ ID NO. 1, and oligonucleotide sequence 2, and the sequence is shown as SEQ ID NO. 2.

[0005] Further, the capture antibodies coupled with the oligonucleotide sequence coat the magnetic beads.

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

[0007] Step 1, raw material preparation: preparing the single molecule immunodetection raw material, comprising:

[0008] Step 1.1, site-specific coupling of antibodies and oligonucleotides;

[0009] Step 1.2, coating magnetic beads with capture antibodies coupled with oligonucleotide sequences;

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

[0011] Step 3, connecting the reaction system to form a rolling circle amplification template

[0012] Step 4, rolling circle amplification for detection signal amplification

[0013] Step 5, fluorescence signal counting.

[0014] Further, step 1.1 is specifically as follows: according to the detection marker, selecting a suitable antibody pair, which is divided into detection antibodies and capture antibodies; the detection antibodies and the capture antibodies are site-specifically coupled with azide functional groups through one-step enzyme catalysis; the detection antibodies and the capture antibodies coupled with sodium azide functional groups are respectively coupled with oligonucleotide sequence 1 and oligonucleotide sequence 2 modified with dibenzocyclooctyne (DBCO), and the coupling mode 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 detected by BCA quantification.

[0015] Further, step 1.2 is specifically as follows: selecting 2-10 μm magnetic beads with amino functional groups labeled with fluorescence markers; coupling the capture antibodies coupled with the oligonucleotide sequences with the above magnetic beads through trans-click chemistry reaction; removing the capture antibodies not coated on the magnetic beads by magnetic bead washing buffer.

[0016] Further, step 2 is specifically as follows:

[0017] The magnetic beads coated with the capture antibody are incubated with the sample at room temperature for 1 hour, liquid is removed, and washing buffer is added for cleaning; the washing buffer is removed, and the detection antibody coupled with the oligonucleotide 1 is added to incubate the magnetic beads coated with the capture antibody, and incubation is performed at room temperature for 1 hour; liquid is removed, and washing buffer is added for cleaning.

[0018] Further, step 3 is specifically as follows:

[0019] A ligation reaction system is configured, containing a ligase, a ligation enzyme, and ligation enzyme working buffer, and is incubated with the magnetic beads of step 2 at room temperature for 30 minutes; the incubation reaction liquid is removed, and washing buffer is added for cleaning.

[0020] Further, step 4, rolling circle amplification, is performed to amplify the detection signal

[0021] A rolling circle amplification reaction system is configured, containing isothermal amplification enzyme, amplification substrate dNTP, amplification reaction buffer Mg2+ and fluorescent signal probe hybridized with the rolling circle amplification template; incubation is performed at room temperature for 30-60 minutes; the incubation reaction liquid is removed, and washing buffer is added for cleaning, the washing buffer is removed, and the magnetic beads of step 3 are resuspended with magnetic bead resuspension buffer.

[0022] Further, step 5 is specifically as follows:

[0023] The magnetic beads obtained in step 4 are added dropwise to a 1cm*1cm glass slide area, the entire area of the added magnetic beads is scanned by a fluorescence scanning microscope, and the positive signal points are counted.

[0024] The present application can realize the detection of a few grams of biomarkers through single molecule immunodetection raw materials and a detection method. The entire detection process can be completed at room temperature in a laboratory without the need for temperature incubation or special equipment, and the results can be interpreted by matching a conventional flow cytometer or a fluorescence microscope. There is no need to customize chips and consumables, and the cost is controllable. The double antibody sandwich method is used, and the detection specificity is higher. The rolling circle amplification signal amplification technology is integrated, and the experimental single molecule signal recognition accuracy is improved.

[0025] The detection method of the present application combines non-site coupling of a capture antibody with solid-phase magnetic beads, and the capture antibody and the detection antibody are coupled with a specific sequence oligonucleotide sequence by one-step enzyme catalysis, and the detection signal is amplified by rolling circle amplification, forming a high-efficiency detection system, which can be used for the detection of protein markers in serum, plasma, tissue fluid, cerebrospinal fluid and the like. Compared with the current single molecule immunodetection method, the present application has the advantages of high sensitivity, high specificity, simple and intuitive detection method, improved stability of the detection system and operation repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a flowchart of the single molecule immunodetection method of the present application. DETAILED DESCRIPTION

[0027] The application will be further described below with reference to the accompanying drawings.

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

[0029] Step 1, raw material preparation:

[0030] Step 1.1 Site-specific coupling of antibody and oligonucleotide

[0031] According to the selection of the detection marker, the appropriate antibody pair is selected, which is divided into detection antibody and capture antibody; the detection antibody and the capture antibody are site-specifically coupled with azide functional groups by one-step enzyme catalysis; the detection antibody and the capture antibody coupled with sodium azide functional groups are respectively coupled with oligonucleotide sequence 1 modified with dibenzocyclooctyne (DBCO), and oligonucleotide sequence 2, the sequence is as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the coupling mode 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 detected by BCA quantification.

[0032] Step 1.2 Capture antibody coupled with oligonucleotide sequence coated magnetic beads

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

[0034] Step 2, configuration of single molecule immune reaction system

[0035] The capture antibody coated magnetic bead raw material is incubated with the sample at room temperature for 1 hour, the liquid is removed, and the washing buffer 2 is added for washing; the washing buffer 2 is removed, and the detection antibody coupled with oligonucleotide 1 is added to incubate with the capture antibody coated magnetic beads, and incubated at room temperature for 1 hour; the liquid is removed, and the washing buffer 2 is added for washing.

[0036] Step 3, connection reaction system to form rolling circle amplification template

[0037] The connection reaction system is configured to contain a linker, a ligase, a ligase working buffer, etc., and is incubated with the magnetic beads of step 2 at room temperature for 30 minutes; the incubation reaction liquid is removed, and the washing buffer 3 is added for washing.

[0038] Step 4, rolling circle amplification for detection signal amplification

[0039] The rolling circle amplification reaction system is configured to include isothermal amplification enzyme, amplification substrate dNTP, amplification reaction buffer Mg2+, and a fluorescent signal probe hybridized with the rolling circle amplification template; incubation is performed at room temperature for 30 minutes to 60 minutes; the incubation reaction liquid is removed, washing buffer 4 is added for washing, the washing buffer 4 is removed, and the magnetic beads in step 3 are resuspended with a magnetic bead resuspension buffer.

[0040] Step 5, fluorescent signal counting

[0041] The magnetic beads obtained in step 4 are added dropwise to a 1cm*1cm glass area, the entire area of the added magnetic beads is scanned by a fluorescence scanning microscope, and the positive signal points are counted.

[0042] Embodiment

[0043] Taking IL-6 detection in a plasma sample as an example, the operation process of the single-molecule immunodetection method of the application is as follows:

[0044] 1. The principle of replacing the original antibody storage solution with a dialysis bag buffer solution is to replace the original antibody storage solution with a 25mM Tris-HCL buffer solution with a pH value of 7.2, and adjust the antibody concentration to 1-10mg / ml with the Tris-HCL buffer solution.

[0045] 2. The total amount of the above-mentioned IL-6 detection antibody 1mg is mixed with Endo-S enzyme according to a mass ratio of 1:5 to 1:20, and the substrate disaccharide sugar oxa (N3-LacNAc-oxa) is mixed with the antibody according to a molar ratio of 10:1 to 50:1, and incubated at room temperature for 30 minutes to 60 minutes, and the proportion of the catalytic combination of the azide group on the sugar chain of the antibody can reach more than 90%. The above-mentioned IL-6 capture antibody is catalytically combined with the azide group on the sugar chain in the same way.

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

[0047] 4. Synthesize oligonucleotide 1 and oligonucleotide 2 containing dibenzocyclooctyne (DBCO) modification at the end, and the sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively; the oligonucleotide is dissolved in 100-200 micromoles of ultrapure water or phosphate buffer. The above-mentioned IL-6 detection antibody prepared in step 3 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; the above-mentioned IL-6 capture antibody is incubated with oligonucleotide sequence 2 in the same way.

[0048] 5. Replace the detection antibody reaction system incubated with oligonucleotide sequence 1 using an ultrafiltration tube or dialysis bag to remove excess free oligonucleotide sequence 1. Replace the coupling buffer with phosphate buffer, maintaining a neutral pH and a phosphate molar concentration of 10 mM. Perform the same steps as for the capture antibody reaction system incubated with oligonucleotide sequence 2.

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

[0050] 7. The tetrazine-NHS compound reacts with the amino groups on the magnetic beads. The ratio of the compound to the magnetic beads is 1 million to 2 million:1. The mixture is incubated at room temperature by rotation for 2 hours. The beads are washed 3 times with washing buffer 2 for 3 minutes each time. The washing buffer 2 is removed by a magnetic rack. The magnetic beads are then re-treated with neutral 10mM phosphate solution.

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

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

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

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

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

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

[0057] 14、The slide is pre-marked with a 1*1 cm area using an immunohistochemical pen. Resuspend the magnetic beads obtained in step 13 above with resuspension buffer and drop them into the area, and scan the target area by a fluorescence scanner.

[0058]

[0059] Table 1 Buffer composition table.

Claims

1. A method for single molecule immunoassay for non-diagnostic purposes, comprising the following steps: Step 1, raw material preparation: preparing single molecule immunoassay raw materials, the single molecule immunoassay raw materials comprising IL-6 detection antibody and IL-6 capture antibody, the detection antibody and the capture antibody being coupled with azide functional groups on the sugar chain by disaccharide sugar oxazoline, the detection antibody and the capture antibody coupled with sodium azide functional groups being coupled with oligonucleotide sequence 1 modified with dibenzocyclooctyne, the sequence being shown as SEQ ID NO. 1, and oligonucleotide sequence 2, the sequence being shown as SEQ ID NO. 2; comprising: Step 1.1, site-specific coupling of antibody and oligonucleotide; Step 1.2, IL-6 capture antibody coupled with oligonucleotide sequence coating magnetic beads; Step 2, configuring a single molecule immune reaction system; Step 3, connecting the reaction system to form a rolling circle amplification template Step 4, rolling circle amplification for detection signal amplification Step 5, fluorescence signal counting; Step 3 is specifically as follows: configuring a ligation reaction system, containing ligase 1 and ligase 2, the sequences being shown as SEQ ID NO. 3 and SEQ ID NO. 4 respectively, ligase, ligase working buffer, incubating the magnetic beads of step 2 at room temperature for 30 minutes; removing the incubation reaction solution, adding washing buffer for cleaning; Step 4 is specifically as follows: configuring a rolling circle amplification reaction system, containing isothermal amplification enzyme, amplification substrate dNTP, amplification reaction buffer Mg2 + and fluorescence signal probe hybridized with the rolling circle amplification template, the sequence of the fluorescence signal probe being shown as SEQ ID NO. 5; incubating at room temperature for 30-60 minutes; removing the incubation reaction solution, adding washing buffer for cleaning, removing the washing buffer, and resuspending the magnetic beads of step 3 with magnetic bead resuspension buffer. 2.The method for single molecule immunoassay for non-diagnostic purposes according to claim 1, wherein: Step 1.1 is specifically as follows: selecting appropriate antibody pairs according to the detection marker, which are IL-6 detection antibody and IL-6 capture antibody; the detection antibody and the capture antibody are site-specifically coupled with azide functional groups by one-step enzyme catalysis; the detection antibody and the capture antibody coupled with sodium azide functional groups are coupled with oligonucleotide sequence 1 and oligonucleotide sequence 2 modified with dibenzocyclooctyne, respectively, and the coupling mode 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 detected by BCA quantification. 3.The method for single molecule immunoassay for non-diagnostic purposes according to claim 1, wherein: Step 1.2 is specifically as follows: selecting 2-10 μm magnetic beads with amino functional groups labeled with fluorescence markers; coupling the IL-6 capture antibody coupled with oligonucleotide sequence with the above magnetic beads through trans-click chemistry reaction; removing the IL-6 capture antibody not coated on the magnetic beads by magnetic bead washing buffer. 4.The method for single molecule immunoassay for non-diagnostic purposes according to claim 1, wherein: Step 2 is specifically as follows: The magnetic beads coated with IL-6 capture antibody are incubated with the sample at room temperature for 1 hour, the liquid is removed, and the washing buffer is added for washing; the washing buffer is removed, and the detection antibody coupled with oligonucleotide 1 is added to incubate with the capture antibody coated magnetic beads, and incubation is performed at room temperature for 1 hour; The liquid is removed, and the washing buffer is added for washing.

5. The single molecule immunodetection method for non-diagnostic purposes according to claim 1, characterized in that: Step 5 is specifically as follows: The magnetic beads obtained in step 4 are dropped on a 1cm*1cm glass slide area, the entire area of the dropped magnetic beads is scanned by a fluorescence scanning microscope, and the positive signal points are counted.

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