Method for improving detection efficiency of proximity ligation technology

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for improving the detection efficiency of an ortho-ligation technique, which comprises the following steps: enriching target molecules in a form of coating an antibody with a microbead, combining with an ortho probe, connecting the ortho probe by using a splint probe, and finally determining the existence and quantity of the target molecules through qPCR (quantitative polymerase chain reaction) detection. The method provided by the invention not only can detect low-abundance target molecules, but also has higher detection accuracy compared with a conventional method.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for improving the detection efficiency of proximity ligation technology. Background Art

[0002] Proximity Ligation Assay (PLA) is a highly sensitive in vitro protein analysis technology based on an enzyme-linked mechanism and a linker (an artificially designed linear oligonucleotide single chain, whose two ends can be complementary to a specific target sequence. When the linker hybridizes with the target sequence, they will be close to each other in space, and the two ends of the probe can be connected into a chain under the action of DNA ligase). This method first uses a pair of proximity probes to dual-recognize the target molecule to generate an amplifiable detection signal, and then realizes timed quantitative detection through quantitative PCR, converting the detection of protein into the detection of DNA, with extremely high sensitivity and specificity. PLA can analyze the signal of low-abundance proteins by amplifying antibody-antigen binding events, making it suitable for the development of multiplex detection for simultaneous quantification of protein and nucleic acid targets.

[0003] PLA detection includes many forms, such as homogeneous assay, solid phase assay, etc. Among them, solid phase PLA detection is favored because it can enrich target molecules with low abundance and eliminate the influence of other substances in complex biological samples on the determination. However, solid phase PLA detection still has the problem that the probe relies on random collision and is difficult to effectively connect in low-abundance targets, resulting in low PLA detection efficiency. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a microbead enrichment PLA assay method. Specifically, the target molecules are enriched in the form of microbeads coated with antibodies. Since each microbead can be loaded with up to hundreds of antibodies, multiple target molecules can be enriched on a single microbead, thereby obtaining a significantly higher degree of enrichment, which facilitates subsequent probe connection and can significantly improve the connection efficiency. In addition, the present invention also found that the use of conventionally designed linkers in this process has the problem of signal loss, which is overcome by using the splint probe designed by the present invention.

[0005] Specifically, the present invention provides a method for improving the detection efficiency of proximity ligation technology, which comprises the following steps: (a) providing a microbead complex, the microbead complex comprising: (i) microbead carrier; (ii) a first antibody that specifically binds to a target molecule and is immobilized on the surface of the microbead; (b) incubating the microbead complex with a sample to be tested, so that the first antibody binds to the target molecule; (c) separating the microbead complex from the sample to be tested to obtain a microbead complex bound to the target molecule; (d) incubating the microbead complex bound to the target molecule with a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a second antibody coupled to the 5' end of the first oligonucleotide, and the second proximity probe comprises a third antibody coupled to the 3' end of the second oligonucleotide, wherein the second antibody and the third antibody each bind to a different epitope on the target molecule or each binds to a different epitope on another target molecule that interacts with the target molecule, thereby obtaining a microbead complex bound to the first proximity probe and the second proximity probe; (e) incubating the microbead complex of step (d) with a splint probe, wherein the splint probe consists of short hairpins located at both ends and a hybridization region located in the middle, wherein the hybridization region contains complementary sequences complementary to the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide, respectively, so that the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide are adjacent to each other by hybridization with the splint probe; (f) incubating the microbead complex of step (e) with DNA ligase, so that the 3' end of the adjacent first oligonucleotide is ligated to the 5' end of the second oligonucleotide; (g) mixing the microbead complex of step (f) with a qPCR reaction system containing primers respectively complementary to the first oligonucleotide and the second oligonucleotide, and performing a qPCR reaction to determine the presence of the target molecule or the two interacting target molecules.

[0006] As used herein, the method of the present invention can be used to detect the presence of a single target molecule or the presence of two interacting target molecules, and those skilled in the art can select and adapt the method of the present invention according to actual needs. For example, in the case of detecting a single target molecule, the first, second, and third antibodies are all able to specifically bind to the target molecule, but each recognizes a different epitope on the target molecule, i.e., has different antigenic determinants from each other. In the case of detecting two interacting target molecules, the first antibody specifically binds to the first target molecule, while the second and third antibodies specifically bind to the second target molecule that interacts with the first target molecule, and the second and third antibodies each recognize a different epitope on the second target molecule, i.e., have different antigenic determinants from each other.

[0007] Furthermore, the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

[0008] Furthermore, the first antibody is fixed on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding.

[0009] Furthermore, the coupling of the second antibody and the third antibody to the oligonucleotide is achieved by chemical cross-linking or biotin-streptavidin binding.

[0010] Furthermore, the chemical cross-linking is selected from EDC / NHS coupling, click chemistry (CuAAC) or maleimide-thiol reaction.

[0011] Furthermore, the antibody may be a monoclonal antibody or a polyclonal antibody.

[0012] Furthermore, the sequence of the first oligonucleotide is shown in SEQ ID NO:1.

[0013] Furthermore, the sequence of the second oligonucleotide is shown in SEQ ID NO:2.

[0014] Furthermore, the splint probe is a dumbbell-shaped oligonucleotide, which comprises short hairpins at both ends and a hybridization region in the middle.

[0015] Furthermore, the short hairpins at both ends of the splint probe each have a loop with a length of 4-6 bases and a reverse complementary double strand with a length of 4-6 base pairs.

[0016] Furthermore, the hybridization region in the middle of the splint probe is single-stranded and has a length of 8-12 bases, wherein the complementary sequence complementary to the 3' end of the first oligonucleotide has a length of 4-6 bases, and the complementary sequence complementary to the 5' end of the second oligonucleotide has a length of 4-6 bases.

[0017] Preferably, the short hairpins at both ends of the splint probe each have a loop with a length of 4 bases and a reverse complementary double strand with a length of 4 base pairs, and the complementary sequence complementary to the 3' end of the first oligonucleotide is 6 bases in length, and the complementary sequence complementary to the 5' end of the second oligonucleotide is 6 bases in length.

[0018] Furthermore, the sequence of the splint probe is shown in SEQ ID NO:3.

[0019] Furthermore, the method may also combine steps eg together, for example, may include incubating the microbead complex of step (d) with a qPCR reaction system comprising a splint probe, DNA ligase and primers and performing a qPCR reaction to determine the presence of the target molecule or two interacting target molecules.

[0020] Furthermore, the DNA ligase is T4 DNA ligase.

[0021] Furthermore, the qPCR reaction system also includes a reaction buffer, Taq DNA polymerase, ATP, dNTP, a detection probe and nuclease-free water.

[0022] Furthermore, both ends of the detection probe are labeled with a fluorescent group and a quenching group, respectively.

[0023] Furthermore, the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5; and the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0024] Furthermore, the detection probe is a Taqman probe.

[0025] Advantageous Effects of the Invention The present invention first improves the existing solid phase PLA assay format by using microbeads coated with a large number of antibodies to highly enrich the target molecules, so that the proximity probes and target molecules can collide and bind more efficiently. As can be seen from the examples, the method of the present invention can detect target molecules as low as 0.001 ng / mL, with excellent detection sensitivity.

[0026] However, during this method, multiple proximity probes will bind to each target molecule on the same microbead at the same time, and the presence of more proximity probes on the same microbead may result in the proximity of the proximity probes of each target molecule, resulting in the number of detected target molecules being lower than the actual number of target molecules. For conventional linkers, since it is necessary to consider the strength and specificity of binding to the template, as well as the length of the duplex that the ligase must meet for the connection, its length is usually longer, reaching about 30bp. However, the present invention finds that when linkers of such length are measured by the microbead enrichment PLA of the present invention, the measured value described above is low. In order to solve this problem, the present invention uses a unique splint probe to replace the conventional linker, and it is found that the presence of the target molecule can be detected more accurately. Compared with conventional linkers, the short hairpin structures at both ends of the splint probe of the present invention allow the stable binding of the ligase and the execution of the connection reaction, so the length is shorter. In addition, the splint probe with a dumbbell-shaped structure has a higher rigidity, the flexibility of the molecular chain is lower, and it is more suitable for the microbead enrichment PLA method of the present invention, reducing the situation of the proximity of the proximity probes of multiple target molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic structural diagram of the clamping probe of the present invention is shown.

[0028] Figure 2The amplification curves of the TNF-α content in the samples determined by microbead-enriched PLA in the example are shown (1 is 1000 ng / mL; 2 is 100 ng / mL; 3 is 10 ng / mL; 4 is 1 ng / mL; 5 is 0.1 ng / mL; 6 is 0.01 ng / mL; 7 is 0.001 ng / mL).

[0029] Figure 3 The Ct value standard curve of the TNF-α content in the sample determined by microbead-enriched PLA in the example is shown.

[0030] Figure 4 The Ct value standard curve of TNF-α content in the microbead-enriched PLA assay sample in the comparative example is shown. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] The antibodies used in the examples were purchased from Suzhou Jinan Protein Technology Co., Ltd., wherein the first antibody was a biotinylated TNF-α antibody (Fabgennix, TNFA-BIOTIN), the second antibody was a TNF-α antibody (Invitrogen, PA5-120124), and the third antibody was a TNF-α antibody (Invitrogen, PA5-19810), wherein the first, second and third antibodies each had a different antigenic determinant; human TNF-α recombinant protein was purchased from MedChemExpress (HY-P700291).

[0033] The sequences of the oligonucleotides, splint probes and primers used in the examples and the conventional linkers used in the comparative examples are shown in Table 1.

[0034] Table 1

[0035] Example: Determination of TNF-α content in samples by microbead-enriched PLA (1) Preparation of microbead complex: Incubate 1 μL Dynabeads™ MyOne™ Streptavidin T1 magnetic beads with 100 μL primary antibody dilution (concentration 100 μg / mL) at room temperature for 30 min under stirring, collect the beads by magnetic separation, wash three times with 10 mM PBS, incubate the beads in 10 mM PBS containing 0.1% BSA with shaking for 15 min, collect the beads and resuspend them in 10 mM PBS; (2) TNF-α protein was dissolved in 10 mM PBS to prepare sample solutions of different concentrations (0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL), and incubated with the microbead complex at room temperature for 1 h. (3) Magnetic separation of the microbead complex, washing three times and resuspending in 50 μL 10 mM PBS; (4) Preparation of proximity probes: 10 μL of 2 μg / μL of the second antibody and the third antibody were added to 1 μL of 4 mM dibenzocyclooctyne-PEG4-N-hydroxysuccinimide ester (DBCO) for reaction at room temperature for 30 minutes to add an NHS group to the N-terminus, and 1 μL of 1 M Tris-HCl was added for incubation at room temperature for 5 minutes to terminate the above reaction; 20 μg of the second antibody and the third antibody modified with the NHS group were incubated with 40 μM of the first oligonucleotide modified with an azide group at the 5' end and the second oligonucleotide modified with an azide group at the 3' end dissolved in 10 mM PBS at 4°C overnight to couple the second antibody to the 5' end of the first oligonucleotide and the third antibody to the 3' end of the second oligonucleotide, thereby obtaining the first and second proximity probes; (5) diluting the first and second proximity probes to 1 μg / mL with PBS, mixing them in equal volumes, and then mixing them with an equal volume of the microbead complex separated in step (3), and incubating them with rotation at room temperature for 90 minutes to obtain an incubation solution; (6) Prepare the PCR reaction system according to Table 2 Table 2:

[0036] (7) Add 5 μL of incubation solution to the PCR reaction system, perform qPCR reaction according to the procedure in Table 3 and output the Ct value. The amplification curve is as follows: Figure 2 As shown, the logarithmic value of TNF-α concentration is used as the x-axis and the Ct value is used as the y-axis to establish a standard curve, as shown in FIG. Figure 3 As shown, the regression equation is Y = -3.393*X + 25.49, and the correlation coefficient R 2 =0.9990.

[0037] Table 3:

[0038] Comparative Example: Determination of TNF-α in samples using conventional linkers in microbead-enriched PLA Steps (1) to (5) are the same as in the embodiment; Step (6): replace the splint probe in Table 2 with the linker, and keep the other components unchanged; Step (7) is the same as in the example, and the relationship between the output Ct value and the TNF-α concentration is as follows: Figure 4 shown.

[0039] from Figure 3 and Figure 4 It can be seen from the comparison that compared with the splint probe used in the present invention, the microbead-enriched PLA using conventional linkers requires a higher Ct value to detect the same concentration of TNF-α, and the correlation coefficient of the standard curve is only 0.9716, indicating that its linear relationship is relatively poor. Figure 4 From the data, it can be found that with the increase of TNF-α concentration, its Ct value shows a downward trend that is first slow and then fast. This also confirms the inventor's speculation that with the increase of adjacent probes bound to the same microbead, the number of adjacent probes on different target molecules crossing and approaching each other will also increase. When the TNF-α concentration is high enough, the signal is strong enough to exceed the detection limit, resulting in little change in the Ct value difference.

[0040] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for improving the detection efficiency of proximity ligation technology, characterized in that: The following steps are involved: (a) providing a microbead complex, the microbead complex comprising: (i) microbead carrier; (ii) a first antibody that specifically binds to a target molecule and is immobilized on the surface of the microbead; (b) incubating the microbead complex with a sample to be tested, so that the first antibody binds to the target molecule; (c) separating the microbead complex from the sample to be tested to obtain a microbead complex bound to the target molecule; (d) incubating the microbead complex bound to the target molecule with a first proximity probe and a second proximity probe, wherein the first proximity probe comprises a second antibody coupled to the 5' end of the first oligonucleotide, and the second proximity probe comprises a third antibody coupled to the 3' end of the second oligonucleotide, wherein the second antibody and the third antibody each bind to a different epitope on the target molecule or each binds to a different epitope on another target molecule that interacts with the target molecule, thereby obtaining a microbead complex bound to the first proximity probe and the second proximity probe; (e) incubating the microbead complex of step (d) with a splint probe, wherein the splint probe consists of short hairpins located at both ends and a hybridization region located in the middle, wherein the hybridization region contains complementary sequences complementary to the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide, respectively, so that the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide are adjacent to each other by hybridization with the splint probe; (f) incubating the microbead complex of step (e) with DNA ligase, so that the 3' end of the adjacent first oligonucleotide is ligated to the 5' end of the second oligonucleotide; (g) mixing the microbead complex of step (f) with a qPCR reaction system containing primers respectively complementary to the first oligonucleotide and the second oligonucleotide, and performing a qPCR reaction to determine the presence of the target molecule or the two interacting target molecules.

2. The method according to claim 1, characterized in that The microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

3. The method according to claim 1, characterized in that The first antibody is immobilized on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding.

4. The method according to claim 1, characterized in that: The coupling of the second and third antibodies to the oligonucleotides is achieved by chemical cross-linking or biotin-streptavidin binding.

5. The method according to claim 4, characterized in that The chemical cross-linking is selected from EDC / NHS coupling, click chemistry (CuAAC) or maleimide-thiol reaction.

6. The method according to claim 1, characterized in that The short hairpins at both ends of the splint probe each have a loop of 4-6 bases in length and a reverse complementary double strand of 4-6 base pairs in length, and the complementary sequences of the hybridization region in the middle of the splint probe that are complementary to the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide are 4-6 bases in length.

7. The method according to claim 1, characterized in that The method comprises incubating the microbead complex of step (d) with a qPCR reaction system comprising a splint probe, a DNA ligase and a primer and performing a qPCR reaction, thereby determining the presence of a target molecule or two interacting target molecules.

8. The method according to claim 7, characterized in that The qPCR reaction system also includes a reaction buffer, TaqDNA polymerase, ATP, dNTP, TaqMan probe and nuclease-free water.

Images