A method for improving the detection efficiency of proximity ligation technology

Abstract

The present invention belongs to the field of biological detection technology, and particularly relates to a method for improving the detection efficiency of proximity ligation technology, which includes enriching target molecules in the form of antibody-coated microbeads, binding them to proximity probes, then using splint probes to ligate the probes in proximity, and finally determining the presence and quantity of target molecules by qPCR detection. The method of the present invention can not only detect low-abundance target molecules, but also has higher detection accuracy compared with conventional methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly 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 technique based on an enzyme-linked mechanism and linkers (an artificially designed linear oligonucleotide single strand, the two ends of which can be complementary to specific target sequences. When the linkers hybridize with the target sequences, they will be close to each other in space, and the two ends of the probes can be ligated into a strand under the action of DNA ligase). This method first uses a pair of proximity probes to double-recognize the target molecule to generate an amplifiable detection signal, and then realizes the timing and quantitative detection through quantitative PCR, converting the detection of proteins into the detection of DNA, with extremely high sensitivity and specificity. PLA can analyze low-abundance proteins by amplifying the signal of antibody-antigen binding events, making it suitable for the development of multiplex assays for simultaneously quantifying protein and nucleic acid targets.

[0003] PLA detection includes various forms, such as homogeneous assays, solid-phase assays, etc. Among them, solid-phase PLA detection is favored because it can enrich target molecules with lower abundance and can exclude the influence of other substances in complex biological samples on the assay. However, solid-phase PLA assays still have the problem that probe-dependent random collisions are difficult to effectively ligate in low-abundance targets, resulting in low PLA detection efficiency. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for microbead-enriched PLA assay. Specifically, the form of microbeads coated with antibodies is used to enrich target molecules. Since each microbead can carry up to hundreds of antibodies, multiple target molecules can be enriched on a single microbead, thus obtaining a significantly higher enrichment degree, which facilitates subsequent probe ligation and can significantly improve the ligation efficiency. In addition, the present invention also discovers that there is a problem of signal loss when using conventionally designed linkers in this process, which is overcome by using the splint probes designed by the present invention.

[0005] Specifically, the present invention provides a method for improving the detection efficiency of proximity ligation technology, which includes the following steps:

[0006] (a) Providing a microbead complex, the microbead complex comprising:

[0007] (i) A microbead carrier;

[0008] (ii) A first antibody specifically binding to the target molecule immobilized on the surface of the microbead;

[0009] (b) Incubate the microbead complex with the sample to be tested so that the first antibody binds to the target molecule;

[0010] (c) Separate the microbead complex from the sample to be tested to obtain a microbead complex bound to the target molecule;

[0011] (d) Incubate the microbead complex bound to the target molecule with a first proximity probe and a second proximity probe. The first proximity probe comprises a second antibody conjugated to the 5'-end of a first oligonucleotide, and the second proximity probe comprises a third antibody conjugated to the 3'-end of a second oligonucleotide. The second antibody and the third antibody each bind to different epitopes on the target molecule or each bind to different epitopes 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;

[0012] (e) Incubate the microbead complex of step (d) with a splint probe, which consists of short hairpins at both ends and a hybridization region in the middle. The hybridization region comprises complementary sequences that are respectively complementary to the 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide, such that the 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide are adjacent to each other by hybridizing with the splint probe;

[0013] (f) Incubate the microbead complex of step (e) with a DNA ligase so that the adjacent 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide are ligated together;

[0014] (g) Mix the microbead complex of step (f) with a qPCR reaction system comprising primers that are respectively complementary to the first oligonucleotide and the second oligonucleotide, and perform a qPCR reaction to determine the presence of the target molecule or two interacting target molecules.

[0015] 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. Those skilled in the art can select according to actual needs and make adaptive adjustments to the method of the present invention. For example, in the case of detecting a single target molecule, the first, second, and third antibodies can specifically bind to the target molecule, but each recognizes a different epitope on the target molecule, that is, they have 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 different epitopes on the second target molecule, that is, they have different antigenic determinants from each other.

[0016] Further, the microbead carrier is a magnetic bead modified with surface carboxylation, amination, or streptavidin.

[0017] Furthermore, the first antibody is immobilized on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding.

[0018] Furthermore, the conjugation of the second antibody and the third antibody with the oligonucleotide is achieved by chemical cross-linking or biotin-streptavidin binding.

[0019] Furthermore, the chemical cross-linking is selected from EDC / NHS conjugation, click chemistry (CuAAC), or maleimide-thiol reaction.

[0020] Furthermore, the antibody can be a monoclonal antibody or a polyclonal antibody.

[0021] Furthermore, the sequence of the first oligonucleotide is as shown in SEQ ID NO: 1.

[0022] Furthermore, the sequence of the second oligonucleotide is as shown in SEQ ID NO: 2.

[0023] Furthermore, the splint probe is a dumbbell-shaped oligonucleotide, which comprises short hairpins at both ends and a hybridization region in the middle.

[0024] Furthermore, each of the short hairpins at both ends of the splint probe has a loop with a length of 4-6 bases and an inverted complementary double strand with a length of 4-6 base pairs.

[0025] Furthermore, the hybridization region in the middle of the splint probe is single-stranded, with a length of 8-12 bases, wherein the length of the complementary sequence complementary to the 3'-end of the first oligonucleotide is 4-6 bases, and the length of the complementary sequence complementary to the 5'-end of the second oligonucleotide is 4-6 bases.

[0026] Preferably, each of the short hairpins at both ends of the splint probe has a loop with a length of 4 bases and an inverted complementary double strand with a length of 4 base pairs, and the length of the complementary sequence complementary to the 3'-end of the first oligonucleotide is 6 bases, and the length of the complementary sequence complementary to the 5'-end of the second oligonucleotide is 6 bases.

[0027] Furthermore, the sequence of the splint probe is as shown in SEQ ID NO: 3.

[0028] Furthermore, the method can also combine steps e-g together. For example, it can include incubating the microbead complex in step (d) with a qPCR reaction system containing a splint probe, a DNA ligase, and a primer and performing a qPCR reaction to determine the presence of a target molecule or two target molecules that interact.

[0029] Furthermore, the DNA ligase is T4 DNA ligase.

[0030] Furthermore, the qPCR reaction system further comprises a reaction buffer, Taq DNA polymerase, ATP, dNTP, a detection probe, and nuclease-free water.

[0031] Furthermore, both ends of the detection probe are respectively labeled with a fluorescent group and a quenching group.

[0032] Furthermore, the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX, and CY5; the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2, and BHQ3.

[0033] Furthermore, the detection probe is a Taqman probe.

[0034] Advantages of the Invention

[0035] 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, the proximity probes and the 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, having excellent detection sensitivity.

[0036] However, during this method process, multiple proximity probes bind to their respective target molecules simultaneously on the same microbead. The presence of a large number of proximity probes on the same microbead may cause the proximity probes of each target molecule to cross and approach each other, resulting in the detected number of target molecules being lower than the actual number of target molecules. For conventional linkers, since they need to consider both the binding strength and specificity with the template and the double-stranded length required for the ligase to perform ligation, their length is usually relatively long, reaching about 30 bp. The present invention has found that when such a length of linker is used in the microbead-enriched PLA assay of the present invention, there will be a situation where the measured value is on the low side as described above. To solve this problem, the present invention uses a unique splint probe to replace the conventional linker and finds that it can more accurately detect the presence of target molecules. Compared with the conventional linker, 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 ligation reaction, so the length is shorter. In addition, the dumbbell-shaped splint probe has higher rigidity and lower flexibility of the molecular chain, which is more suitable for the microbead-enriched PLA method of the present invention and reduces the situation where the proximity probes of multiple target molecules cross and approach each other. Brief Description of the Drawings

[0037] Figure 1 Shows a schematic structural diagram of the splint probe of the present invention.

[0038] Figure 2Shows the amplification curves of the content of TNF-α in the sample determined by bead-enriched PLA in the examples (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).

[0039] Figure 3 Shows the standard curve of Ct values for the content of TNF-α in the sample determined by bead-enriched PLA in the examples.

[0040] Figure 4 Shows the standard curve of Ct values for the content of TNF-α in the sample determined by bead-enriched PLA in the comparative examples. Detailed implementation mode

[0041] The present invention will be further described below in conjunction with specific embodiments, but the embodiments 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 technical field.

[0042] The antibodies used in the examples were purchased from Novoprotein Scientific Inc., Suzhou. Among them, the first antibody is a biotinylated TNF-α antibody (Fabgennix, TNFA-BIOTIN), the second antibody is a TNF-α antibody (Invitrogen, PA5-120124), and the third antibody is a TNF-α antibody (Invitrogen, PA5-19810). The first, second and third antibodies each have different antigenic determinants; the human TNF-α recombinant protein was purchased from MedChemExpress (HY-P700291).

[0043] The sequences of the oligonucleotides, splint probes and primers used in the examples and the conventional linker used in the comparative examples are shown in Table 1.

[0044] Table 1

[0045]

[0046] Example: Determination of the content of TNF-α in the sample by bead-enriched PLA

[0047] (1) Preparation of bead complexes: 1 μL of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads was incubated with 100 μL of the first antibody dilution solution (concentration: 100 μg / mL) at room temperature for 30 min under stirring. The magnetic beads were collected by magnetic separation, washed 3 times with 10 mM PBS, and the magnetic beads were incubated by shaking in 10 mM PBS containing 0.1% BSA for 15 minutes. The magnetic beads were collected and resuspended in 10 mM PBS;

[0048] (2) Dissolve TNF-α protein in 10 mM PBS to prepare sample solutions with 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 incubate them with the microbead complex at room temperature for 1 h respectively;

[0049] (3) Magnetically separate the microbead complex, wash it 3 times and then resuspend it in 50 μL of 10 mM PBS;

[0050] (4) Prepare the proximity probes: Take 10 μL of the second antibody and the third antibody at 2 μg / μL respectively, add 1 μL of 4 mM dibenzocyclooctyne-PEG4-N-hydroxysuccinimide ester (DBCO), react at room temperature for 30 minutes to add an NHS group to its N-terminus, and add 1 μL of 1 M Tris-HCl and incubate at room temperature for 5 minutes to terminate the above reaction; Incubate 20 μg of the second antibody and the third antibody modified with the NHS group 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, so that the second antibody is conjugated to the 5'-end of the first oligonucleotide and the third antibody is conjugated to the 3'-end of the second oligonucleotide to obtain the first and second proximity probes;

[0051] (5) Dilute the first and second proximity probes to 1 μg / mL with PBS, mix them evenly in equal volumes, and then mix them with the microbead complex separated in step (3) in equal volumes, and incubate with rotation at room temperature for 90 min to obtain an incubation solution;

[0052] (6) Prepare the PCR reaction system according to Table 2

[0053] Table 2:

[0054]

[0055] (7) Add 5 μL of the incubation solution to the PCR reaction system in step, perform qPCR reaction according to the program in Table 3 and output the Ct value. Its amplification curve is as Figure 2 shown. Take the logarithm of the TNF-α concentration as the x-axis and the Ct value as the y-axis to establish a standard curve, as Figure 3 shown. The regression equation is Y = -3.393*X + 25.49, and the correlation coefficient R 2 = 0.9990.

[0056] Table 3:

[0057]

[0058] Comparative example: The content of TNF-α in the sample was determined by microbead enrichment PLA using a conventional linker.

[0059] Steps (1)-(5) were the same as those in the examples.

[0060] Step (6): Replace the splint probe in Table 2 with a linker, and keep the other components unchanged.

[0061] Step (7) was the same as that in the examples. The relationship between the output Ct value and the TNF-α concentration was as Figure 4 shown.

[0062] From Figure 3 and Figure 4 comparison, it can be seen that compared with the splint probe used in the present invention, microbead enrichment PLA using a conventional linker 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. Further analysis of Figure 4 the data shows that as the concentration of TNF-α increases, its Ct value shows a trend of first decreasing slowly and then rapidly, which also confirms the inventor's speculation that as the number of adjacent probes bound to the same microbead increases, the situation of adjacent probes on different target molecules approaching each other crosswise will also increase. When the concentration of TNF-α is high enough, due to the strong signal exceeding the detection limit, the difference in Ct values changes little.

[0063] It should be noted that the present invention has been described with reference to the preferred embodiments in the specification and its drawings. 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 additional limitations to 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. Further, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as within the scope described in the specification of the present invention. Further, for those of ordinary skill in the art, improvements or changes can be made according to the above description, and all such improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for improving the detection efficiency of proximity ligation technology, characterized in that, Comprising the following steps: (a) Providing a microbead complex, the microbead complex comprising: (i) A microbead carrier; (ii) A first antibody that specifically binds a target molecule and is immobilized on the surface of the microbead; (b) Incubating the microbead complex with a sample to be tested such 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, the first proximity probe comprising a second antibody conjugated to the 5'-end of a first oligonucleotide, the second proximity probe comprising a third antibody conjugated to the 3'-end of a second oligonucleotide, the second antibody and the third antibody each binding to a different epitope on the target molecule or each binding 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; Incubating the microbead complex with a qPCR reaction system comprising a splint probe, a DNA ligase, and a primer and performing a qPCR reaction to thereby determine the presence of the target molecule or two interacting target molecules; (e) Incubating the microbead complex of step (d) with a splint probe, the splint probe consisting of hairpins at both ends and a hybridization region in the middle, the hybridization region comprising complementary sequences that are respectively complementary to the 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide, such that the 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide are adjacent to each other by hybridizing with the splint probe; the sequence of the first oligonucleotide is as shown in SEQ ID NO: 1, the sequence of the second oligonucleotide is as shown in SEQ ID NO: 2, and the sequence of the splint probe is as shown in SEQ ID NO: 3; The hairpins at both ends of the splint probe each have a loop with a length of 4-6 bases and an inverted complementary double strand with a length of 4-6 base pairs, and the complementary sequences in the hybridization region in the middle of the splint probe that are respectively complementary to the 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide each have a length of 4-6 bases; (f) Incubating the microbead complex of step (e) with a DNA ligase such that the adjacent 3'-end of the first oligonucleotide and the 5'-end of the second oligonucleotide are ligated together; (g) Mixing the microbead complex of step (f) with a qPCR reaction system comprising primers that are respectively complementary to the first oligonucleotide and the second oligonucleotide and performing a qPCR reaction to thereby determine the presence of the target molecule or two interacting target molecules.

2. The method according to claim 1, wherein The microbead carrier is a magnetic bead surface-modified with carboxyl, amino, or streptavidin.

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

4. The method according to claim 1, wherein The conjugation of the second antibody and the third antibody to the oligonucleotide is achieved by chemical crosslinking or biotin-streptavidin binding.

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

6. The method according to claim 5, wherein The qPCR reaction system further comprises a reaction buffer, Taq DNA polymerase, ATP, dNTP, TaqMan probe and nuclease-free water.

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