A method for simultaneous detection of multiple targets based on proximity ligation

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a method for simultaneous detection of multiple targets based on proximity ligation, comprising using multiple microbeads coated with antibodies that recognize different targets to simultaneously enrich multiple target molecules, and combining them with proximity probes, followed by using splint probes to connect the probes in the proximity position, and finally achieving detection of multiple different target molecules through qPCR detection; the method of the present invention can not only simultaneously detect multiple different target molecules, but also has higher detection accuracy than conventional methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a method for simultaneous detection of multiple targets based on proximity ligation. Background Art

[0002] Proximity Ligation Assay (PLA) is a highly sensitive in vitro protein analysis technique based on an enzyme-linked mechanism and a linker (a single, artificially designed linear oligonucleotide whose ends are complementary to a specific target sequence. When the linker hybridizes to the target sequence, they become spatially close, allowing the two ends of the probe to join into a chain under the action of DNA ligase). This method first utilizes a pair of proximity probes to dually recognize the target molecule, generating an amplifiable detection signal. Quantitative PCR is then used to achieve timed, quantitative detection, converting protein detection into DNA detection, resulting in extremely high sensitivity and specificity. PLA can analyze low-abundance proteins by amplifying the signal from antibody-antigen binding events, making it suitable for the development of multiplexed assays for the simultaneous quantification of protein and nucleic acid targets.

[0003] PLA assays include various formats, such as homogeneous and solid-phase assays. Solid-phase PLA assays are popular because they can enrich low-abundance target molecules and eliminate the influence of other substances in complex biological samples. However, solid-phase PLA assays still suffer from the problem of probe reliance on random collisions, making it difficult to effectively connect to low-abundance targets, resulting in low PLA assay efficiency.

[0004] Sensitive, specific, and multiplexed detection of target molecules has always been a core issue in analytical science, particularly in biomedical research, early disease diagnosis, drug screening, and environmental monitoring. In clinical diagnosis, the onset and progression of many diseases are often accompanied by abnormal expression of multiple biomarkers. Therefore, developing a method that can simultaneously, accurately, and quantitatively detect multiple targets is crucial for early disease screening, typing diagnosis, and personalized treatment. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for simultaneous detection of multiple targets based on microbead enrichment PLA assay. 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. On this basis, the present invention uses a variety of microbeads coated with antibodies that recognize different targets to simultaneously enrich multiple target molecules, thereby achieving simultaneous detection of multiple targets. 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.

[0006] Specifically, the present invention provides a method for simultaneous detection of multiple targets based on proximity ligation, comprising the following steps:

[0007] (a) providing a plurality of microbead complexes, each microbead complex comprising a microbead carrier and a first antibody immobilized on the surface of the microbead carrier, wherein the first antibodies specifically bind to different target molecules;

[0008] (b) incubating the plurality of microbead complexes with a sample to be tested, allowing the first antibodies to bind to their respective target molecules;

[0009] (c) The incubated complexes are separated to obtain a variety of microbead complexes bound to different target molecules.

[0010] Furthermore, the first antibody is fixed on the surface of the microbead carrier by chemical cross-linking or biotin-streptavidin binding, and the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

[0011] The method further comprises:

[0012] (d) incubating the plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the first proximity probe comprises a second antibody coupled to the 5' end of a first oligonucleotide, and the second proximity probe comprises a third antibody coupled to the 3' end of a second oligonucleotide, wherein the second and third antibodies respectively bind to different epitopes of their respective target molecules.

[0013] Furthermore, the first oligonucleotide and the second oligonucleotide comprise a unified first adapter sequence and a unified second adapter sequence at the 3' end and the 5' end, respectively, and the remaining sequences of the oligonucleotides in different probe pairs are different.

[0014] Furthermore, the coupling of the second antibody and the third antibody to the oligonucleotide is achieved by EDC / NHS coupling, click chemistry or maleimide-thiol reaction.

[0015] The method further comprises:

[0016] (e) incubating the proximity probe-bound microbead complex with a splint probe, wherein the splint probe comprises short hairpins at both ends and a hybridization region in the middle, wherein the hybridization region comprises sequences complementary to the first adaptor sequence and the second adaptor sequence, respectively, such that the first and second oligonucleotides are adjacent to each other.

[0017] 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.

[0018] The method further comprises:

[0019] (f) Incubating the multiple microbead complexes obtained in step S5 with DNA ligase, so that the adjacent 3' ends of the first oligonucleotides are ligated to the 5' ends of the second oligonucleotides to form complete ligation products.

[0020] The method further comprises:

[0021] (g) mixing the complex obtained in step (f) with a qPCR reaction system containing a plurality of primer pairs complementary to the first and second oligonucleotides in each probe and performing amplification detection.

[0022] Furthermore, the qPCR reaction system includes Taq DNA polymerase, buffer, ATP, dNTP, multiple detection probes and nuclease-free water. The two ends of the detection probe are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups of each probe are different to distinguish the target molecules.

[0023] In addition, the present invention also provides a method for simultaneous detection of multiple targets based on proximity ligation, which comprises the following steps:

[0024] (a) providing a plurality of microbead complexes, each of the plurality of microbead complexes comprising:

[0025] (i) microbead carriers; and

[0026] (ii) a first antibody that specifically binds to a target molecule and is immobilized on the surface of the microbead carrier;

[0027] wherein the first antibodies immobilized on different microbead complexes each specifically bind to a different target molecule;

[0028] (b) incubating the multiple microbead complexes with a sample to be tested, so that the first antibodies immobilized on different microbead complexes bind to their respective target molecules;

[0029] (c) separating the multiple microbead complexes from the sample to be tested to obtain multiple microbead complexes bound to different target molecules;

[0030] (d) incubating a plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the plurality of first proximity probes and second proximity probe pairs correspond to different target molecules, respectively, wherein the first proximity probe comprises a second antibody coupled to the 5' end of a first oligonucleotide, the second proximity probe comprises a third antibody coupled to the 3' end of a second oligonucleotide, the sequences of the first oligonucleotide and the second oligonucleotide in each pair of first proximity probes and second proximity probes are different among the plurality of first proximity probes and second proximity probe pairs, but all first oligonucleotides have the same first adaptor sequence at their 3' ends and all second oligonucleotides have the same second adaptor sequence at their 5' ends, and the second antibody and the third antibody in each pair of first proximity probes and second proximity probes each bind to a different epitope on the target molecule corresponding to the first proximity probe and second proximity probe pair, thereby obtaining a plurality of microbead complexes bound to the plurality of first proximity probes and second proximity probe pairs;

[0031] (e) incubating the plurality of microbead complexes of step (d) with a splint probe, wherein the splint probe comprises short hairpins at both ends and a hybridization region in the middle, wherein the hybridization region comprises complementary sequences complementary to the first adapter sequence and the second adapter sequence, respectively, such 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;

[0032] (f) incubating the plurality of microbead complexes from step (e) with DNA ligase, such that the adjacent 3' ends of the first oligonucleotides are ligated to the 5' ends of the second oligonucleotides;

[0033] (g) mixing the plurality of microbead complexes of step (f) with a qPCR reaction system comprising a plurality of primer pairs, and performing a qPCR reaction to determine the presence of the target molecule or two interacting target molecules, wherein the plurality of primer pairs correspond to a plurality of first proximity probes and second proximity probe pairs, respectively, and each pair of primers comprises an upstream primer and a downstream primer that are each complementary to the first oligonucleotide and the second oligonucleotide in the corresponding first proximity probe and second proximity probe pair.

[0034] As used herein, a plurality of microbead complexes may include at least two microbead complexes that specifically recognize different target molecules, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more microbead complexes. The microbeads of each microbead complex are immobilized with a first antibody that specifically binds to the same target molecule, i.e., each microbead complex specifically recognizes the same target molecule, and each microbead complex may contain at least one or more microbead complexes that specifically recognize the same target molecule, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more microbead complexes.

[0035] Furthermore, the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

[0036] Furthermore, the first antibody is immobilized on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding.

[0037] Furthermore, the coupling of the second antibody and the third antibody to the oligonucleotide is achieved by chemical cross-linking or biotin-streptavidin binding.

[0038] Furthermore, the chemical cross-linking is selected from EDC / NHS coupling, click chemistry (CuAAC) or maleimide-thiol reaction.

[0039] Furthermore, the antibody may be a monoclonal antibody or a polyclonal antibody.

[0040] As used herein, the first antibody of each microbead complex and the second and third antibodies of each corresponding first proximity probe and second proximity probe pair can specifically bind to the same target molecule, but each recognizes a different epitope on the target molecule, i.e., has different antigenic determinants from each other.

[0041] Furthermore, the splint probe is a dumbbell-shaped oligonucleotide comprising short hairpins at both ends and a hybridization region in the middle.

[0042] 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.

[0043] 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 first adapter sequence has a length of 4-6 bases, and the complementary sequence complementary to the second adapter sequence has a length of 4-6 bases.

[0044] 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 length of the complementary sequence complementary to the first adapter sequence is 6 bases, and the length of the complementary sequence complementary to the second adapter sequence is 6 bases.

[0045] Furthermore, the method may also combine steps eg together, for example, it may include incubating the multiple microbead complexes of step (d) with a qPCR reaction system comprising a splint probe, DNA ligase, and multiple primer pairs and performing a qPCR reaction to determine the presence of multiple different target molecules.

[0046] Furthermore, the DNA ligase is T4 DNA ligase.

[0047] Furthermore, the qPCR reaction system further comprises a reaction buffer, Taq DNA polymerase, ATP, dNTP, a plurality of detection probes and nuclease-free water.

[0048] Furthermore, each of the plurality of detection probes is used to detect a different target molecule, for example, is complementary to the first or second oligonucleotide in a plurality of first proximity probe and second proximity probe pairs corresponding to different target molecules.

[0049] Furthermore, both ends of the multiple detection probes are labeled with a fluorescent group and a quenching group, respectively, and the fluorescent groups of the multiple detection probes are different from each other.

[0050] Furthermore, the fluorescent group may be selected from but not limited to FAM, HEX, VIC, TET, ROX, CY5, etc.; the quenching group may include at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0051] Furthermore, the detection probe is a Taqman probe.

[0052] Advantageous Effects of the Invention

[0053] This invention first improves upon existing solid-phase PLA assay formats by utilizing microbeads coated with a large number of antibodies to highly enrich target molecules, enabling more efficient collision and binding between proximity probes and target molecules. As demonstrated in the examples, the method can detect target molecules as low as 0.001 ng / mL, demonstrating excellent sensitivity.

[0054] However, during this method, multiple proximity probes will bind to each target molecule simultaneously on the same microbead. The presence of more proximity probes on the same microbead may result in the proximity of the proximity probes of each target molecule crossing and approaching each other, resulting in the number of detected target molecules being lower than the actual number of target molecules.

[0055] 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 to perform the connection, its length is usually longer, reaching about 30bp. The present invention found that when a linker of such a length is used in the microbead enrichment PLA assay of the present invention, the measurement 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 splint probe of the present invention has a short hairpin structure at both ends that allows the ligase to bind stably and perform the connection reaction, so it is shorter in length. In addition, the dumbbell-shaped splint probe has a higher rigidity and a lower flexibility of the molecular chain, which is more suitable for the microbead enrichment PLA method of the present invention, reducing the cross-approach between adjacent probes of multiple target molecules.

[0056] On this basis, in order to achieve simultaneous detection of multiple targets, the present invention will simultaneously incubate the sample to be tested with microbead complexes that recognize different target molecules, and improve the sequence of the adjacent probe pairs used to introduce a universal adapter sequence, so that a single splint probe can be used to achieve simultaneous detection of multiple different targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 A schematic structural diagram of the splint probe of the present invention is shown.

[0058] Figure 2 The amplification curve of the TNF-α content in the sample was shown in the embodiment by microbead-enriched multiplex PLA assay.

[0059] Figure 3 The amplification curve of the IL-6 content in the sample of the microbead-enriched multiplex PLA assay in the example is shown.

[0060] Figure 4 The figure shows the Ct value standard curve of the microbead-enriched multiple PLA assay for TNF-α content in samples in the example.

[0061] Figure 5 The figure shows the Ct value standard curve of the IL-6 content in the samples of the microbead-enriched multiplex PLA assay in the example.

[0062] Figure 6 The figure shows the Ct value standard curve of TNF-α content in samples determined by microbead-enriched multiple PLA in the comparative example.

[0063] Figure 7 The figure shows the Ct value standard curve of IL-6 content in samples of microbead-enriched multiple PLA assay in the comparative example. DETAILED DESCRIPTION

[0064] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0065] The first antibody against TNF-α used in the examples 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); the first antibody against IL-6 was a biotinylated IL-6 antibody (Invitrogen, 13-7068-81), the second antibody was an IL-6 antibody (Invitrogen, MA5-44642), and the third antibody was an IL-6 antibody (Invitrogen, 701028), wherein the first, second, and third antibodies each had a different antigenic determinant; human TNF-α recombinant protein was purchased from MedChemExpress (HY-P700291); and human IL-6 recombinant protein was purchased from Gibco (PHC0061).

[0066] 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.

[0067] Table 1: Sequences of oligonucleotides, splint probes and primers, and conventional linkers of comparative examples

[0068]

[0069] Example: Determination of TNF-α and IL-6 in samples by microbead-enriched multiplex PLA

[0070] (1) Prepare a mixture of microbead complexes targeting TNF-α and IL-6: incubate 1 μL of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads with 100 μL of TNF-α or IL-6 primary antibody dilution (concentration of 100 μg / mL) at room temperature for 30 min under stirring, collect the magnetic beads by magnetic separation, wash them three times with 10 mM PBS, and incubate the magnetic beads in 10 mM PBS containing 0.1% BSA for 15 min with shaking. Collect the magnetic beads targeting TNF-α and IL-6, mix them together, and resuspend them in 10 mM PBS;

[0071] (2) TNF-α and IL-6 proteins were dissolved in 10 mM PBS to prepare mixed sample solutions of different concentrations (0 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1000 ng / mL, respectively), and incubated with the microbead complex mixture at room temperature for 1 h.

[0072] (3) Magnetic separation of the microbead complex, washing three times, and resuspending in 50 μL 10 mM PBS;

[0073] (4) Preparation of proximity probes for TNF-α and IL-6: 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) and reacted at room temperature for 30 minutes to add an NHS group to the N-terminus. 1 μL of 1 M Tris-HCl was added and incubated 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.

[0074] (5) Dilute the first and second proximity probes targeting TNF-α and IL-6, respectively, to 1 μg / mL with PBS, mix the four probe solutions in equal volumes, and then mix them with equal volumes of the microbead complex separated in step (3), and incubate them with rotation at room temperature for 90 min to obtain an incubation solution;

[0075] (6) Prepare the PCR reaction system according to Table 2

[0076] Table 2: PCR reaction system

[0077]

[0078] (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 curves of TNF-α and IL-6 (three repeated experiments) are as follows: Figure 2 and 3 As shown, the linear analysis is as follows Figure 4 and 5 shown.

[0079] Table 3: qPCR reaction procedure

[0080]

[0081] Comparative Example: Determination of TNF-α and IL-6 in samples using conventional linkers in microbead-enriched multiplex PLA

[0082] Steps (1) to (5) are the same as in the embodiment;

[0083] Step (6): Replace the splint probe in Table 2 with a conventional linker, and keep the rest of the components unchanged;

[0084] Step (7) is the same as in the embodiment, detecting the relationship between the output Ct value and concentration of TNF-α and IL-6 ( Figure 3 ) found that compared with the splint probe used in the present invention, the linear relationship of microbead-enriched PLA multiplex target detection using conventional linkers was poor, and a higher Ct value was required to detect the same concentration of target molecules, indicating that the detection accuracy using conventional linkers was poor.

[0085] 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 intended to be 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 can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for simultaneous detection of multiple targets based on proximity ligation, characterized in that: The following steps are involved: (a) providing a plurality of microbead complexes, each of the plurality of microbead complexes comprising: (i) microbead carriers; and (ii) a first antibody that specifically binds to a target molecule and is immobilized on the surface of the microbead carrier; wherein the first antibodies immobilized on different microbead complexes each specifically bind to a different target molecule; (b) incubating the multiple microbead complexes with a sample to be tested, so that the first antibodies immobilized on different microbead complexes bind to their respective target molecules; (c) separating the multiple microbead complexes from the sample to be tested to obtain multiple microbead complexes bound to different target molecules; (d) incubating a plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the plurality of first proximity probes and second proximity probe pairs correspond to different target molecules, respectively, wherein the first proximity probe comprises a second antibody coupled to the 5' end of a first oligonucleotide, the second proximity probe comprises a third antibody coupled to the 3' end of a second oligonucleotide, the sequences of the first oligonucleotide and the second oligonucleotide in each pair of first proximity probes and second proximity probes are different among the plurality of first proximity probes and second proximity probe pairs, but all first oligonucleotides have the same first adaptor sequence at their 3' ends and all second oligonucleotides have the same second adaptor sequence at their 5' ends, and the second antibody and the third antibody in each pair of first proximity probes and second proximity probes each bind to a different epitope on the target molecule corresponding to the first proximity probe and second proximity probe pair, thereby obtaining a plurality of microbead complexes bound to the plurality of first proximity probes and second proximity probe pairs; (e) incubating the plurality of microbead complexes of step (d) with a splint probe, wherein the splint probe comprises short hairpins at both ends and a hybridization region in the middle, wherein the hybridization region comprises complementary sequences complementary to the first adapter sequence and the second adapter sequence, respectively, such 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; 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; 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 first adapter sequence has a length of 4-6 bases, and the complementary sequence complementary to the second adapter sequence has a length of 4-6 bases; (f) incubating the plurality of microbead complexes from step (e) with DNA ligase, such that the adjacent 3' ends of the first oligonucleotides are ligated to the 5' ends of the second oligonucleotides; (g) mixing the multiple microbead complexes from step (f) with a qPCR reaction system containing multiple primer pairs, 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 a surface carboxylation, aminoation or streptavidin modification; the multiple primer pairs correspond to multiple first proximity probes and second proximity probe pairs, and each pair of primers consists of an upstream primer and a downstream primer that are complementary to the first oligonucleotide and the second oligonucleotide in the corresponding first proximity probe and the second proximity probe pair.

3. The method according to claim 2, characterized in that The first antibody is immobilized on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding; The coupling of the second and third antibodies to the oligonucleotides is achieved by chemical cross-linking or biotin-streptavidin binding.

4. The method according to claim 3, characterized in that The chemical cross-linking is selected from EDC / NHS coupling, click chemistry or maleimide-thiol reaction.

5. The method according to claim 4, characterized in that The method comprises incubating the multiple microbead complexes of step (d) with a qPCR reaction system comprising a splint probe, DNA ligase, and multiple primer pairs and performing a qPCR reaction, thereby determining the presence of multiple different target molecules.

6. The method according to claim 5, characterized in that The qPCR reaction system further comprises a reaction buffer, TaqDNA polymerase, ATP, dNTP, a plurality of detection probes and nuclease-free water.

7. The method according to claim 6, characterized in that Both ends of the multiple detection probes are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups of the multiple detection probes are different from each other.

Images