CN117867070A

CN117867070A - Detection method and kit

Info

Publication number: CN117867070A
Application number: CN202410058493.8A
Authority: CN
Inventors: 邵伟; 贾蕊; 田雨; 许俊泉
Original assignee: Gewu Intelligent Manufacturing Technology Chengdu Co ltd; Gewu Zhihe Biotechnology Beijing Co ltd
Current assignee: Gewu Intelligent Manufacturing Technology Chengdu Co ltd; Gewu Zhihe Biotechnology Beijing Co ltd
Priority date: 2024-01-15
Filing date: 2024-01-15
Publication date: 2024-04-12

Abstract

The disclosure provides a detection method, which sequentially comprises the following steps: (1) The method comprises the following steps of sequentially carrying out the following steps on more than two samples to be tested: (1-1) mixing a first antibody linked to a first probe, a second antibody linked to a second probe, a sample discrimination sequence and a sample to be tested in a solution to obtain an immune complex; (1-2) contacting the solid surface with the solution obtained in step (1-1) to capture the immune complex to the solid surface; (1-3) washing the solid surface more than once to remove molecules not bound to the solid surface; (2) combining solids for two or more samples to be tested; (3) Adding a connection system to enable immune complexes bound on the solid surface to generate ortho-connection reaction, and optionally purifying ortho-connection reaction products; (4) Eluting, releasing immune complexes bound on the solid surface into a first elution buffer; and (5) detection. Correspondingly, the disclosure also provides a kit.

Description

Detection method and kit

Technical Field

The present disclosure relates to the field of molecular biology, and more particularly to an improved detection method and kit.

Background

In various biological samples (such as serum, plasma, body fluid and the like) or cell lysate, detection and quantification of micro-analytes (such as disease diagnosis marker proteins and drug effect indicating proteins) are very important for early screening of diseases, prediction of occurrence and development of various diseases, improvement of clinical diagnosis accuracy, research of disease occurrence and development mechanism and research and judgment of drug treatment effects.

Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the gold standards for rapid and accurate quantification of proteins. The enrichment of the target protein by the capture antibody and the signal amplification of the detection antibody enable the sandwich ELISA reaction to rapidly, accurately and quantitatively determine the target protein with low background. The main disadvantage of this method is that its detection is limited to nanograms per milliliter and low concentrations of protein cannot be detected. For example, early cancer, alzheimer's detection, etc. require effective detection of analytes at fg/mL concentrations, which is beyond the capabilities of ELISA immunoassay techniques. Meanwhile, for the traditional sandwich ELISA reaction, one reaction system can only detect one protein; if multiple proteins are to be detected, multiple reaction wells are required, which greatly increases the amount of sample used, thus increasing the experimental effort and cost of detection.

In 2002, the ortholigation technique (PLA) has received a great deal of attention due to its high sensitivity and specificity, which is an important complement to traditional immunization methods. The proximity ligation technique is to recognize the object to be detected by using 2 kinds of antibodies marked with nucleic acid sequences, fix the modified nucleic acid sequences at the proximity after the antibodies are combined with the object to be detected, ligate the nucleic acid sequences under the action of nucleic acid ligase, and perform subsequent nucleic acid amplification by using the ligated nucleic acid sequences as templates. In short, the proximity ligation technique achieves high specificity through recognition of various antibodies and high sensitivity through nucleic acid amplification. The approach of proximity ligation assays differs from other in situ hybridization applications based on antigen-antibody reactions in that it uses two antibodies (rather than a single antibody) to recognize different epitopes of the same protein, thereby increasing protein specificity. Furthermore, the use of two antibodies to recognize different proteins can both reveal the location and quantify protein interactions. The advantage that the proximity ligation reaction can only occur when a pair of probes are both bound to the target protein and sufficiently close to each other, and thus the non-specific detection of PLA is very small, results in PLA having higher specificity and detection sensitivity compared to conventional PCR or RCA methods. At endogenous expression levels of the protein of interest, signal enhancement of the detection step provides unique capabilities for studying stable and transient interactions. PLA is highly suitable for high throughput cells and screening experiments as a basis, in conjunction with various image processing and analysis systems.

Briefly, PLA technology achieves high specificity through a variety of antibody recognition and high sensitivity through nucleic acid amplification. The sensitivity of PLA technology is higher than that of traditional ELISA measurement, and can effectively detect early cancers, alzheimer's disease and other diseases needing to be standardized by fg/mL concentration.

Conventional orthographic ligation techniques include homogeneous orthographic ligation techniques (also known as liquid orthographic ligation techniques) and solid orthographic ligation techniques. The homogeneous phase/liquid phase ortho-position connection technology has simple steps, and all experimental steps are carried out in the same solution. However, the background of homogeneous/liquid phase ortho-ligation technology is relatively high due to the lack of a washing step; meanwhile, since the nucleic acid ligase is easily inactivated in complex biological samples (including serum or plasma), the reaction efficiency is affected, resulting in reduced sensitivity. Solid phase proximity ligation techniques, such as nucleic acid-ligated immuno-sandwich assay (NULISA) techniques, employ two magnetic beads, two capture sequences, each capturing an "antigen-antibody" complex; the washing step reduces background interference, and the subsequent qPCR or second generation sequencing can realize single detection/co-detection of the protein. However, this method requires two types of magnetic beads, two capture sequences, and the experimental cost is high; meanwhile, during most of the reaction time, all antigen-antibody complexes are captured on the surface of the magnetic beads, and when the concentration of the protein to be detected is high or a plurality of proteins are detected together, incorrect connection of antibody coupling sequences is easy to occur, so that the duty ratio of useful data is reduced. Furthermore, the current solid orthographic ligation technique requires multiple washing procedures to be performed and each sample requires a separate reaction: for example, 96 samples are processed to obtain 96 products, and for units such as a detection center which need to process a large amount of samples every day, if no specially optimized automatic workstation or high-throughput equipment such as an automatic plate washer is adopted, the problems of huge workload, serious manpower consumption and large data quality exist.

In order to solve the above technical problems, the present disclosure provides a technical route such that samples can be combined for subsequent operations in the middle of the reaction. For example, the initial 96 samples can be pooled into 1 sample for manipulation, and this improvement would greatly reduce the difficulty and effort of the experimental manipulation while the data quality is not affected compared to the reaction alone. Therefore, the technology of the present disclosure has strong practicability compared to conventional ELISA, conventional PLA technology, and the like.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present disclosure aims to provide a technical route that can combine samples for subsequent operations in the middle of the reaction of multiple samples, thereby greatly reducing the difficulty and workload of experimental operations, while the quality of data is not affected compared with the single reaction.

In a first aspect, the present disclosure provides a detection method comprising the following steps in order:

(1) The method comprises the following steps of sequentially carrying out the following steps on more than two samples to be tested: (1-1) mixing a first antibody linked to a first probe, a second antibody linked to a second probe, a sample discrimination sequence, and a sample to be tested in a solution (with the aim of binding the first antibody, the second antibody, and the sample discrimination sequence to an analyte in the sample to be tested) to obtain an immunocomplex; (1-2) contacting the solid surface with the solution obtained in step (1-1) to capture the immune complex to the solid surface; (1-3) washing the solid surface more than once to remove molecules not bound to the solid surface;

(2) Combining solids for more than two samples to be tested;

(3) Adding a connection system to enable immune complexes bound on the solid surface to generate ortho-connection reaction, and optionally purifying ortho-connection reaction products;

(4) Eluting, releasing immune complexes bound on the solid surface into a first elution buffer; and

(5) Detecting to obtain a detection result of an analyte in a sample to be detected;

in some embodiments, the analyte is one or more modified or unmodified proteins.

In some embodiments, the attachment of the probe to the antibody may be a direct attachment, e.g., the probe has a group that is coupled to the antibody such that the probe is directly coupled to the antibody. In some embodiments, the attachment of the probe to the antibody may be via an indirect attachment of an intermediate molecule (e.g., a linker scaffold), e.g., the antibody is conjugated to the linker scaffold, while the probe has a region that interacts with the linker scaffold.

In some embodiments, the method further comprises, between steps (4) and (5), the steps of: (4' -1) secondary capturing and cleaning: capturing the immune complex on the solid surface again, cleaning the solid surface for more than one time, and removing molecules which are not bound to the solid surface; (4' -2) eluting, releasing the immunocomplexes bound on the solid surface into a second elution buffer.

In some embodiments, in step (5), the detecting comprises fluorescent quantitative PCR (e.g., multiplex fluorescent quantitative PCR) or second generation sequencing. Optionally, the PCR amplification product is purified.

Herein, contacting a solid surface with a solution includes cases where the solid is completely immersed in the solution, where the solid is surrounded by the solution, where one or more surfaces of the solid are in contact with the solution, where one or more surfaces of the solid are immersed by the solution, and so on.

In some embodiments, washing the solid surface more than once includes washing 1-5 times, 1-4 times, 1-3 times, e.g., 1 time, 2 times, 3 times, 4 times, and/or 5 times.

In some embodiments, more than one step/sub-step herein may be repeated 2 times, 3 times, 4 times, 5 times, or more as practical.

In some embodiments, the first antibody and/or the second antibody has a parking group, the solid surface has a capture group, the parking group is bound to the capture group, thereby capturing the immune complex to the solid surface.

In some embodiments, the first probe and/or the second probe has a parking group, the solid surface has a capture group, the parking group is bound to the capture group, thereby capturing the immunocomplexes to the solid surface.

In some embodiments, the immunocomplexes can be captured to the solid surface by a capture sequence that is complementary to the capture region of the first probe and/or the second probe, the capture sequence having a parking group, the solid surface having a capture group, the parking group being bound to the capture group, thereby capturing the immunocomplexes to the solid surface.

In some embodiments, the anchoring group is selected from more than one of biotin, polydT, and polydA.

In some embodiments, the capture group is selected from more than one of streptavidin, avidin, polydT, and polydA.

In some embodiments, the immunocomplexes are captured to the solid surface using a capture sequence that is complementary to the capture region of the first probe and/or the second probe, the capture sequence having a parking group, the solid surface having a capture group, the parking group being bound to the capture group, thereby capturing the immunocomplexes to the solid surface.

In some embodiments, the capture groups of the solid surface in step (1-2) are the same as the capture groups of the solid surface in step (4' -1).

In some embodiments, the capture groups of the solid surface in step (1-2) are different from the capture groups of the solid surface in step (4' -1).

In some embodiments, the parking group is biotin and the capture group of the solid surface is streptavidin and/or avidin.

In some embodiments, the parking group is polydT and the capturing group is polydA.

In some embodiments, the parking group is polydA and the capturing group is polydT.

In some embodiments, the docking group is polydA and biotin and the capturing group is polydT or streptavidin. For example, the capturing group on the solid surface in the step (1-2) is poly dT, and the capturing group on the solid surface in the step (4' -1) is streptavidin.

In some embodiments, the immune complex may also be captured to the solid surface by a thioester group, disulfide bond or cleavable bond (photocleavable, chemically cleavable or enzymatically cleavable bond), protein-protein interaction between the docking group and the capture group of the solid surface.

In some embodiments, binding or release of antibodies to or from a solid surface may be achieved by changing conditions such as salt ion concentration and/or temperature.

In some embodiments, capture and release of immune complexes may be achieved by high salt capture, low salt release methods. For example, capture of the immunocomplexes can be achieved by binding between biotin conjugated to the probe and streptavidin or avidin coupled to the solid surface.

In some embodiments, the ligation system comprises a splint sequence and a ligase. In some embodiments, the ligase is selected from the group consisting of T4 DNA ligase, T7 DNA ligase, splintR ligase, taq ligase. In some embodiments, the ligation system further comprises a ligase buffer.

In some embodiments, the first antibody is coupled to a linker scaffold comprising a region that is compatible with the first probe, a region that is compatible with the sample discrimination sequence, and a 3' terminal modifying group coupled to the first antibody.

In some embodiments, the second antibody is coupled to a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled to the second antibody.

In some embodiments, the first antibody is coupled to a linker scaffold comprising a region that is compatible with the first probe, a region that is compatible with the sample discrimination sequence, and a 5' terminal modifying group coupled to the first antibody.

In some embodiments, the second antibody is coupled to a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 3' terminal modifying group coupled to the second antibody.

In some embodiments, the first antibody is coupled to a linker scaffold comprising a region that is interworked with the first probe, a region that is interworked with the sample discrimination sequence, and a 3' terminal modifying group coupled to the first antibody; and/or the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled with the second antibody.

In some embodiments, the first antibody is coupled to a linker scaffold comprising a region that is interworked with the first probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled to the first antibody; and/or the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 3' terminal modifying group coupled with the second antibody.

Preferably, the 3 'terminal or 5' terminal modifying group is selected from one or more of azide group, amino group and mercapto group.

In some embodiments, the first probe and the second probe each independently comprise an oligonucleotide strand comprising a second generation sequencing linker region, a barcode region, and a splint sequence complementary region. In some embodiments, the oligonucleotide strand further comprises a UMI region and/or a capture region.

In some embodiments, the first probe further comprises a region that interacts with a linker scaffold, and the second probe further comprises an antibody-coupled group at the 3 'end and a phosphorylated group at the 5' end.

In some embodiments, the second probe further comprises a region that interacts with a linker scaffold, and the first probe further comprises a group at the 5' end that is coupled to the antibody.

In some embodiments, the first probe further comprises a region that interacts with a linker scaffold, and the second probe further comprises a group at the 5' end that is coupled to an antibody.

In some embodiments, the second probe further comprises a region that interacts with a linker scaffold, and the first probe further comprises an antibody-coupled group at the 3 'end and a phosphorylated group at the 5' end.

In some embodiments, the first probe and the second probe further comprise regions that interact with a linker scaffold.

In some embodiments, the group coupled to the antibody is selected from more than one of an azide group, an amino group, and a thiol group.

In some embodiments, the first probe comprises a region that interacts with a linker scaffold and a 5' terminal phosphorylating group; and/or the second probe comprises a region that interacts with the linker scaffold and a 5' terminal phosphorylating group.

In some embodiments, the linker scaffold sequence and the first probe are coupled to a first antibody.

In some embodiments, the linker scaffold sequence and the second probe are coupled to a second antibody.

In some embodiments, the linker scaffold sequence and the first probe are coupled to a first antibody and the linker scaffold sequence and the second probe are coupled to a second antibody.

In some embodiments, the sample discrimination sequence comprises a region that interacts with a linker scaffold, a sample barcode region, and a second generation sequencing linker region.

In some embodiments, the proximity ligation reaction product is a nucleic acid reporter comprising a sample discrimination sequence, a UMI region of the first probe and the second probe, a barcode region, and/or a splint sequence complementary region.

In some embodiments, the immunocomplexes obtained after the ligation reaction may be re-enriched with a solid surface and eluted with an elution buffer as a reporter molecule. In some embodiments, the ligated immune complexes are used directly as a reporter or after dilution in a certain proportion.

In some embodiments, in step (1), the solution is a binding buffer selected from one or more of SSC buffer, tris, HEPES, bis-Tris and MOPS. In some embodiments, the cation concentration of the binding buffer is 100mM to 1M, e.g., 100mM to 200mM, 100mM to 400mM, 100mM to 600mM, 100mM to 800mM, 100mM to 1000mM, 200mM to 400mM, 200mM to 600mM, 200mM to 800mM, 200mM to 1000mM, 400mM to 600mM, 400mM to 800mM, 400mM to 1000mM, 500mM to 750mM, 500mM to 1000mM, 600mM to 800mM, 600mM to 1000mM, or 800mM to 1000mM. In some embodiments, the cation is sodium.

In some embodiments, in step (1-3) and step (4' -1), washing is performed using a wash solution selected from more than one of SSC buffer, tris, HEPES, bis-Tris, and MOPS. In some embodiments, the cation concentration of the wash solution is 10mM to 200mM, e.g., 10mM to 50mM,10mM to 100mM, 10mM to 150mM, 20mM to 50mM, 20mM to 100mM, 20mM to 150mM, 20mM to 200mM, 30mM to 50mM, 30mM to 100mM, 30mM to 150mM, 30mM to 200mM, 40mM to 50mM, 40mM to 100mM, 40mM to 150mM, 40mM to 200mM, 50mM to 100mM, 50mM to 150mM, 50mM to 200mM, 60mM to 100mM, 60mM to 150mM, 60mM to 200mM, 70mM to 100mM, 70mM to 200mM, 80mM to 100mM, 80mM to 200mM, 90mM to 100mM, 90mM to 150mM, 100mM to 200mM, 110mM to 150mM, 110mM to 200mM, 120mM to 150mM, 120mM to 200mM, 130mM to 150mM, or 140mM to 150 mM. In some embodiments, the cation is sodium.

In some embodiments, the first elution buffer is selected from more than one of TE buffer, tris, HEPES, bis-Tris, and MOPS. In some embodiments, the cation concentration of the first elution buffer is 1mM to 100mM, e.g., 1mM to 5mM, 5mM to 10mM, 1mM to 10mM, 5mM to 20mM, 5mM to 30mM, 10mM to 50mM, 10mM to 60mM, 10mM to 80mM, 20mM to 40mM, 20mM to 50mM, 20mM to 60mM, 20mM to 80mM, 30mM to 50mM, 30mM to 70mM, 40mM to 60mM, 40mM to 80mM, or 50mM to 100mM. In some embodiments, the cation is sodium.

In some embodiments, the second elution buffer is selected from more than one of DEPC water, RNase/DNase free water, TE buffer, tris, HEPES, bis-Tris, and MOPS. In some embodiments, the second elution buffer is selected from the group consisting of TE buffer, tris, HEPES, bis-Tris, and MOPS, and the cation concentration of the second elution buffer is 1mM to 100mM, e.g., 1mM to 5mM, 5mM to 10mM, 1mM to 10mM, 5mM to 20mM, 5mM to 30mM, 10mM to 50mM, 10mM to 60mM, 10mM to 80mM, 20mM to 40mM, 20mM to 50mM, 20mM to 60mM, 20mM to 80mM, 30mM to 50mM, 30mM to 70mM, 40mM to 60mM, 40mM to 80mM, or 50mM to 100mM. In some embodiments, the cation is sodium.

In some embodiments, the above parameters (e.g., biological sample diluent, binding buffer, elution buffer, wash solution, incubation conditions, splint sequence, ligase species, ligation system, temperature and time, etc.) can be optimally adjusted for the protein detected.

In some embodiments, the solid is selected from the group consisting of magnetic solids, such as magnetic beads, e.g., oligo-dT magnetic beads and SA magnetic beads. In some embodiments, multiple bead types, capture sequences, and modification types may be used in combination. In some embodiments, oligo-dT magnetic beads are used paired with PolyA capture sequences. In some embodiments, the SA magnetic beads are paired with a biotin-modified capture sequence.

In a second aspect, the present disclosure also provides a kit for use in the methods of the present disclosure, comprising:

(i) A first probe and a first antibody;

(ii) A second probe and a second antibody;

(iii) Sample discrimination sequences;

(iv) A binding buffer selected from one or more of SSC buffer, tris, HEPES, bis-Tris and MOPS, preferably having a cation concentration of 100mM to 1M; preferably, the cation is sodium ion;

(v) A wash buffer selected from one or more of SSC buffer, tris, HEPES, bis-Tris and MOPS, preferably the cation concentration of the wash solution is 10mM to 200mM, preferably the cation is sodium ion.

(vi) A first elution buffer selected from one or more of TE buffer, tris, HEPES, bis-Tris and MOPS, preferably the cation concentration of the first elution buffer is 1mM to 100mM, preferably the cation is sodium ion;

(vii) A splint sequence and a ligase; preferably, the ligase is selected from the group consisting of T4 DNA ligase, T7 DNA ligase, splingR ligase, taq ligase;

(viii) An optional capture sequence complementary to the capture region of the first probe and/or the second probe, the capture sequence having a docking group, preferably the docking group being selected from more than one of biotin, polydT, polydA;

(ix) An optional solid; preferably, the solid is selected from magnetic solids such as magnetic beads; and/or preferably, the solid surface has a capture group capable of binding to a parking group, the capture group being selected from more than one of streptavidin, avidin, polydA, polydT sequences.

(x) The optional second elution buffer is selected from more than one of DEPC water, RNase/DNase-free water, TE buffer, tris, HEPES, bis-Tris and MOPS; and

(xi) Alternative PCR amplification reagents.

In some embodiments, the first antibody is coupled to a linker scaffold comprising a region that is interworked with the first probe, a region that is interworked with the sample discrimination sequence, and a 3' terminal modifying group coupled to the first antibody; the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled with the second antibody.

Any combination of the features described herein can be made unless otherwise indicated herein or otherwise clearly contradicted by context. Any feature or combination of features described for the method of the first aspect herein is also applicable to the kit of the second aspect herein unless otherwise indicated herein or clearly contradicted by context.

Compared with the conventional solid surface ortho-position connection technology, the method can combine samples in the middle stage of the reaction of multiple samples, and perform subsequent operation, so that the experimental operation difficulty and workload are greatly reduced, and meanwhile, the quality of data is not affected compared with that of single reaction.

Drawings

Fig. 1 shows a schematic diagram according to an exemplary embodiment of the present disclosure. A: for each sample, the antigen (i.e., analyte in the sample to be tested), the probe, and the antibody (the first antibody is coupled with the linker scaffold, and forms a first probe, the linker scaffold is coupled with the antibody duplex structure, wherein the sample discrimination sequence is complementarily bound to the scaffold sequence, and the second antibody is coupled with the second probe) and the sample discrimination sequence. B: for each sample, the antigen-antibody immune complexes are each bound to a solid (e.g., magnetic bead) surface by a capture sequence. C: combining the samples, and carrying out ortho-ligation under the action of ligase and a splint sequence; d: immune complexes are released from the solid surface and amplified by PCR.

FIG. 2 shows a data comparison (single antigen, single antibody pair) of IL-8 and IL-6 antigen gradients detected singly using a conventional solid phase PLA method, the presently disclosed PLA method (non-pooled samples).

Fig. 3 shows the results of detection using the presently disclosed PLA method (not pooled samples) in the presence of first probe interference. "non-interfering group signal" means that no interfering first probe is added, the detected sample signal is sequenced, "interfering group normal signal" means that the detected sample signal is sequenced in the presence of first probe interference, and "interfering group interfering signal" means that the detected interfering signal is sequenced in the presence of first probe interference.

Fig. 4 shows the results of detection using the presently disclosed PLA method (non-pooled samples) in the presence of pooled sample sequence interference. "non-interfering group signal" means that no interfering sample pooled sequences are added, the detected sample signal is sequenced, "interfering group normal signal" means that the detected sample signal is sequenced in the presence of an interfering sample pooled sequence, and "interfering group interfering signal" means that the detected interference is sequenced in the presence of an interfering sample pooled sequence.

Fig. 5 shows a comparison of data (single antigen, single antibody pair) under an uncombined sample, pooled sample using the presently disclosed PLA method, wherein ■ represents an uncombined sample and ∈ represents a pooled sample.

For purposes of clarity, not necessarily drawn to scale, the various features or elements in the figures may be drawn to scale.

Detailed Description

The specific embodiments presented herein are intended to describe the present disclosure by way of example and are not intended to limit the disclosure in any way, including but not limited to the specific embodiments described herein.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms include the plural and plural terms shall include the singular. In general, the terms used in molecular biology, immunology, genetics, protein, nucleic acid chemistry and hybridization described herein are those commonly used as known to those skilled in the art.

As used herein, "comprising," "including," "having," and the like are intended to be open ended, meaning that there are additional, non-listed elements, components, or steps in addition to the listed elements, components, or steps, which do not materially affect the basic novel characteristics of the methods or products of this disclosure.

As used herein, the term "and/or" as used in the phrase "a and/or B" is intended to include: both A and B, A or B, (separate) A and (separate) B. Likewise, the term "and/or" as used in the phrase "A, B and/or C" is intended to encompass the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; (separate) A; (separate) B; and (separate) C.

As used herein, "first," "second," "third," etc. are used merely to distinguish between structures or elements mentioned in this disclosure and are not intended to limit the position, importance, etc. of each structure or element.

As used herein, "above" and "below" include the present numbers. For example, "more than one" means "one or more", wherein "multiple" means "two or more".

As used herein, unless expressly specified otherwise, ranges of values provided herein are understood to encompass the endpoints of the ranges, each intermediate value, and one tenth of the unit of the lower limit. Unless specifically stated otherwise, ranges excluding one or both of the endpoints are also included within the scope of the disclosure.

Definition of the definition

As used herein, the term "detect" or similar expression is used broadly to include any manner of determining the presence (whether or not present) of an analyte or any form of measurement of the analyte. Thus, "detecting" may include determining, measuring or evaluating the presence or absence or quantity or location of an analyte, including quantitative, semi-quantitative, and qualitative determinations, measurements or evaluations. Such determination, measurement or evaluation may be relative (e.g., when detecting more than two different analytes in a sample), or may be absolute. The term "quantitative" when used in the context of the quantification of a target analyte in a sample may refer to absolute or relative quantification. Absolute quantification may be achieved by including more than one control analyte at known concentrations and/or by comparing the detected target analyte level to known control analytes (e.g., generated by a standard curve). Alternatively, relative quantification may be achieved by comparison of the levels or amounts detected between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.

As used herein, the term "analyte" may be any substance (e.g., molecule) or entity that is detected by the assay methods provided herein. Analytes are targets of the assay methods provided herein. Thus, the analyte may be any biological molecule or compound that needs to be detected, such as a peptide or protein, a nucleic acid molecule, or a small molecule, including organic and inorganic molecules. The analyte may be a cell or microorganism (including a virus) or a fragment or product thereof. In some embodiments, the analyte is a protein or polypeptide. The target analyte includes a protein molecule, such as a polypeptide, protein, or prion, or any molecule that contains a protein or polypeptide component or fragment thereof. In some embodiments, the analyte is all or part of a protein molecule. The analyte may also be a single molecule or a complex containing more than two molecular subunits, which may or may not be covalently bound to each other and may be the same or different. Thus, the analyte that can be detected by the assay methods described herein can be a complex analyte, which can be a protein complex. Aggregates of molecules (e.g., proteins) may also be analytes of interest. The aggregate analytes may be aggregates of the same protein or different proteins. The analyte may also be a complex consisting of a protein or peptide or nucleic acid molecule (e.g., DNA or RNA). In some embodiments, the analyte is a complex composed of both a protein and a nucleic acid, such as a regulatory factor, e.g., a transcription factor. Preferably, the analyte is one or more modified or unmodified proteins.

In this context, the terms "analyte" and "antigen" may be used interchangeably unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "sample" may be any biological and clinical sample, including, for example, any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples, such as cell cultures, cell preparations, cell lysates, and the like. Environmental samples, such as soil and water samples or food samples, are also included. The sample may be freshly prepared or previously processed in any suitable manner (e.g., for storage). Thus, representative samples include any material containing biomolecules, or any other desired analyte or target analyte, including, for example, food and similar products, clinical and environmental samples. The sample may be a biological sample, including viral or cellular material, including prokaryotic or eukaryotic cells, viruses, phages, mycoplasma, protoplasts, and organelles. These biological materials include all types of mammalian and non-mammalian cells, plant cells, algae, including blue-green algae, fungi, bacteria, protozoa, and the like. Representative samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, feces, cerebrospinal fluid or any other bodily fluid (e.g., respiratory secretions, saliva, milk, etc.), tissues, biopsies, cell cultures, cell suspensions, conditioned media or other cell culture component samples, and the like. The sample may be pre-treated in any suitable or desired manner to prepare for use in the methods disclosed herein. For example, the sample may be processed by cell lysis or purification, isolation, etc. of the analyte. For example, neurological biomarkers (e.g., p-Tau181, p-Tau 217) and other disease-related biomarkers in cerebrospinal fluid, serum, or plasma can be detected using the methods of the present disclosure, providing a basis for earlier diagnosis and monitoring of disease; measuring the level of C-reactive protein (CRP) in serum to assist in the differential diagnosis of bacterial infection and non-bacterial infection; in disease plasma and normal plasma samples, multiple protein expression (thousands of protein expression) is detected, and biomarker proteins capable of predicting disease progress are found and used for disease diagnosis and drug efficacy judgment.

As used herein, the term "binding" refers to the interaction between molecules (e.g., antibodies and analytes, or a parking group and a capture group) that form a complex. The interactions may be, for example, non-covalent interactions including hydrogen bonding, ionic bonding, hydrophobic interactions, and/or van der Waals interactions. In some embodiments, the antibody specifically binds to its target analyte, i.e., the antibody binds to the target analyte with greater affinity than other components in the sample. In some embodiments, the binding of the antibody to the target analyte may be different from the binding to the non-target analyte, wherein the antibody does not bind to the non-target analyte or binds to the non-target analyte negligibly or undetectably, or any such non-specific binding (if present) is at a relatively low level that can be distinguished. The binding between the target analyte and its antibody is typically non-covalent. Antibodies used in the methods provided herein can be covalently conjugated to a docking group (e.g., polydA, polyd, biotin, etc.) without significantly eliminating the binding affinity of the antibody to its target analyte.

As used herein, the term "antibody" includes antibody binding fragments or derivatives or mimetics of antibodies, wherein the fragments, derivatives and mimetics have binding affinity for the target analyte. For example, antibody fragments (such as Fv, F (ab) 2 and Fab) can be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Also of interest are recombinantly or synthetically produced antibody fragments or derivatives, such as single chain antibodies or scFv, or other antibody derivatives such as chimeric antibodies or CDR-grafted antibodies, wherein such recombinantly or synthetically produced antibody fragments retain the above The binding characteristics of antibodies, i.e., their ability to specifically bind to the target analyte. Such antibody fragments or derivatives generally include at least V of the antibodies of the disclosure _H And V _L Domains to retain the binding characteristics of the antibodies of the disclosure. The antibody fragments, derivatives or mimetics of the present disclosure can be readily prepared using any convenient methodology. Antibodies and fragments, derivatives, and mimetics thereof can be obtained from commercial sources and/or prepared using any convenient technique. Methods for producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives and mimetics of antibodies (including recombinant derivatives of such antibodies) are known to those of skill in the art.

As used herein, the terms "first antibody" and "second antibody" generally refer to an antibody pair that is capable of specifically binding to the same analyte and thus being spatially proximate to each other, thereby producing a nucleic acid reporter molecule useful for detection of said analyte via the respective coupled first and second probes. In the method provided in the present disclosure, 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25) antibodies may be used simultaneously to detect different analytes in a sample, and the upper limit of the number of types of antibody pairs used simultaneously is not particularly limited, and the number of types of antibody pairs used may be determined according to the specific detection purpose. Wherein each "antibody pair" recognizes an analyte (i.e., each of the 1 or more antibody pairs recognizes a different species of analyte, respectively), preferably each "antibody pair" comprises a barcode (barcode) corresponding to the antibody in the coupled first and/or second probe. For example, in embodiments of the present disclosure, IL-8, IL-6, and PSA in a sample may be detected simultaneously using a "pair of antibodies that recognize IL-8, IL-6, and PSA, respectively.

As used herein, "probe" generally refers to a single-stranded DNA or RNA fragment (about 20 to 500 bp). In this context, detection of the analyte can be achieved by the probe. The skilled artisan can suitably design the corresponding probes for a specific application purpose (e.g., different samples, different analytes, etc.) using methods conventional in the art (e.g., using Primer 3 software, etc.).

As used herein, a "linker scaffold" generally refers to a nucleotide sequence that hybridizes to the free terminal nucleotide sequence of a sample discrimination sequence, the free terminal nucleotide sequence of a probe, and has a group (e.g., azide group, amino group, thiol group, etc.) that can be coupled to an antibody, for forming a complex with the sample discrimination sequence, probe, and antibody.

As used herein, "sample discrimination sequence" refers to an oligonucleotide strand for identifying and discriminating between different samples, the oligonucleotide strand comprising a second generation sequencing linker region, a barcode region, and a linker scaffold complementary region; wherein the second generation sequencing adapter region is one of the subsequent PCR primer binding regions; the bar code area is a section of nucleotide sequence designed for distinguishing each detection sample, and the sequencing data after sample combination can judge which detection sample a certain sequencing signal comes from according to the section of sequence; the splice holder complementary region mates with the free portion of the splice holder.

As used herein, the term "solid" in reference to a "solid surface" generally refers to any solid support suitable for attachment to a nucleic acid or protein and which facilitates the detection step. Examples of solid matrices include particulate materials (e.g., magnetic beads, gel beads), colloids, single interfaces, tubes, chips, multi-well plates, microtiter plates, membranes, gels, and resins. Exemplary solid matrices may include magnetic beads and microtiter plates. When the solid matrix is a particulate material (e.g., magnetic beads), they may be distributed in wells of a multi-well plate or in centrifuge tubes for batch parallel processing. In certain embodiments, the solid surface has capture groups bound to parking groups.

As used herein, the term "capture sequence" generally refers to a nucleotide sequence capable of hybridizing to a capture region of a first probe and/or a second probe and capable of binding to a solid surface, thereby capturing an immune complex described herein to the solid surface. In certain embodiments, the capture sequence has a docking group that binds to a capture group on the solid surface. In certain embodiments of the methods of the present disclosure, the capture sequence may be added in step (1) or in step (2).

As used herein, the terms "parking group" and "capture group" generally refer to any pair of compound molecules capable of coupling to an antibody, probe, capture sequence, etc. herein and capable of binding to each other. In this context, the immunocomplexes described herein are captured to a solid surface (in other words, the immunocomplexes described herein are tethered to a solid surface) by the combination of a "docking group" and a "capture group". The "parking group" and "capture group" may be any pair of compound molecules known in the art that are in accordance with the needs of the present disclosure, such as biotin/streptavidin systems, biotin/avidin systems, poly dT/poly dA systems, and the like.

As used herein, the term "splint sequence" generally refers to a nucleotide sequence that hybridizes to both the free terminal nucleotide sequence of a first probe and the free terminal nucleotide sequence of a second probe. After binding of the splint sequence to the first and second probes, ligation of the first and second probes in the proximity of the first probe may be achieved by the action of a ligase or polymerase. For example, the splint sequence is complementary to nucleotide sequences on the first and second probes that bind the same analyte, forming a partially double stranded DNA structure with gaps that are filled in by the action of a ligase to form a specific nucleic acid reporter.

As used herein, the term "hybridization" refers to the process of binding two nucleic acid strands and forming antiparallel double strands by hydrogen bonding between residues of the two nucleic acid strands. In terms of polynucleotides, "hybridization" and "binding" are used interchangeably.

As used herein, a "primer" is an oligonucleotide, at least a portion of which is complementary to a portion of the sequence of a nucleic acid template to be amplified or replicated. Primers are commonly used in Polymerase Chain Reaction (PCR).

As used herein, the term "complementary" refers to a sufficient number of complementary base pair nucleotides that specifically bind to a target nucleic acid sequence for amplification or detection.

As used herein, the term "splint sequence complementary region" refers to a nucleotide sequence in the first probe and/or the second probe that is capable of hybridizing to a splint sequence. Ligation is initiated by hybridization of the region of sequence complementarity to the splint sequence.

As used herein, the term "capture region" refers to a nucleotide sequence in the first probe and/or the second probe that is capable of hybridizing to a capture sequence. In certain embodiments, the first antibody, the second antibody, the first probe, and/or the second probe have no parking group thereon, the first probe and/or the second probe have a capture region, and the immunocomplexes are captured to the solid surface by hybridization of the capture region in the first probe and/or the second probe to the capture sequence and binding of the parking group of the capture sequence to the capture group of the solid surface.

In this context, the term "molecular tag (Unique Molecular Identifier, UMI)" generally refers to a random nucleotide sequence of individual individuals for distinguishing the same analyte (e.g., antigen) in a sample. The number of starting molecules of the analyte can be more precisely quantified by UMI, and the non-uniformity caused by PCR amplification can be reduced. UMI is typically composed of a random sequence (e.g., NNNNNN, NNNNNNN, NNNNNNNN) or degenerate base (NNNRNYN) of about 6nt to 10 nt.

As used herein, the term "barcode" in the context of reference to "probes" refers to a stretch of nucleotide sequences designed on each probe to effect detection of a different analyte (e.g., antigen) as an identification of the different antibodies to which the probes are coupled, thereby obtaining information about the analyte (e.g., antigen) to which the antibodies bind. In the context of "sample discrimination sequences," the term "barcode" refers to a sequence of nucleotides corresponding to each sample in order to effect the combined detection of different samples, from which the combined sequencing data of the samples can determine from which detected sample a sequencing signal originated.

Examples

Reagents and nucleic acid sequences used in the examples of the present disclosure are shown below:

SSC buffer: 1 XSSC and 0.5 XSSC buffers were prepared with 20 XSSC buffer (pH 7.0, sterile), the 20 XSSC buffer consisting of 3M NaCl and 0.3M sodium citrate.

Capture sequence (sequence number 41 in table 2): at a concentration of 1. Mu.M

Salmon sperm DNA:10mg/mL

Oligo (dT) magnetic beads: 10mg/mL

Splint sequence: number 44 in Table 2 at a concentration of 1. Mu.M

Sample discrimination sequences (sequence numbers 9-40): at a concentration of 200nM

Table 1: probe sequences conjugated to antibodies (lowercase bar code, N molecular tag)

Table 2: capture sequence L, upstream primer and downstream primer, lowercase barcode, N molecular tag

/>

The above-described nucleic acid sequences used in the examples of the present disclosure were synthesized by the division of biological engineering (Shanghai).

In embodiments of the present disclosure, sequencing down machine data is extracted by UMI-tools software from UMI-containing sequences and each antibody-conjugated barcode sequence, and the extracted sequences are counted against UMI by python script.

Example 1: conjugation of probes to antibodies

In the examples section, the inventors validated the methods of the present disclosure using IL-8 (interleukin 8; ebosin) and IL-6 (interleukin 6; ebosin) as examples. In this example, an exemplary group for coupling the linker scaffold sequence and the second probe to the antibody is an Azide group (Azide), and the linker scaffold sequence and the second probe are coupled to the first antibody and the second antibody, respectively, using the DBCO method. For each analyte, a primary antibody and a secondary antibody are used in concert, which recognize different sites of the same analyte (e.g., protein) separately.

For the conventional PLA scheme, a first antibody of IL-8 and IL-6 is coupled to a corresponding first probe sequence and a second antibody is coupled to a corresponding second probe sequence.

The procedure for coupling the probe to the antibody is summarized as follows:

1) The antibody preservation buffer was replaced with PBS buffer, and the antibody concentration was diluted to 2mg/mL with PBS buffer. Antibodies 1 and 2 to IL-8 and IL-6 were both from Eboson.

2) 2mg/mL antibody was used with 2mM DBCO (dibenzocyclooctyne) at a volume ratio of 10:1 are mixed and reacted on ice for 1 hour to prepare a DBCO-labeled antibody.

3) The DBCO labeled antibody was subjected to ultrafiltration purification by an ultrafiltration centrifuge tube, and the concentration of the antibody was measured.

4) The DBCO-labeled antibodies were each reacted with a synthetic single-stranded probe (as shown in table 1) end-modified with an Azide group (Azide) in a molar ratio of 5:1, and incubated overnight at 4 ℃. The probe-conjugated antibodies are obtained by reacting DBCO with an azide group, linking the probe and the antibody together.

5) And (3) carrying out ultrafiltration purification on the single-chain probe coupled antibody, measuring the concentration, and diluting to 20 fmol/mu L for later use.

Example 2: single detection antigen IL-8 or IL-6 (Single antigen, single antibody pair, samples were not pooled) using the PLA method of the present disclosure (multiple libraries were prepared separately)

In this example, IL-8 or IL-6 was used as antigen to be analyzed, and the presently disclosed PLA method (multi-library prepared separately) was used for gradient detection of single antigen, single antibody pairs, samples were not pooled, and the relevant steps were summarized as follows:

1. preparing a first probe and a connector bracket coupled antibody double-chain structure: adding 0.2 mu L of the antibody after coupling the linker scaffold corresponding to the antigen in example 1 and 0.2 mu L of the first probe (L5) into 48.1 mu L of 1 XSSC binding buffer solution, and uniformly mixing; incubating for 10min with shaking at 37deg.C on a thermostatted shaker to allow the first probe to form a double-stranded structure with the adaptor scaffold sequence.

2. Antigen dilution: IL-8 or IL-6 antigen was diluted to the desired concentration using 1 XSSC binding buffer (0.05% Tween 20,0.2% BSA) in a total volume of 50. Mu.L with a maximum antigen concentration of 100pg/mL, 10-fold each dilution yielding a total of 3 antigen concentrations: 100. 10 and 1pg/mL.

3. Antigen-antibody incubation: 0.2. Mu.L of the capture sequence R, 0.1. Mu.L of salmon sperm DNA, 0.2. Mu.L of the first probe of the corresponding antigen formed in step 1, the linker-scaffold conjugated antibody double-stranded structure, 0.2. Mu.L of the second probe conjugated antibody of the corresponding antigen prepared in example 1, 1. Mu.L of the sample discrimination sequence (SEQ ID NO. 9), 50. Mu.L of IL-8 or IL-6 antigen gradient were added to 1 XSSC binding buffer, mixed uniformly, and incubated with shaking at 37℃for 1 hour on a thermostatic shaker, so that the antibody forms an immune complex with the antigen.

4. Immunocomplexes are captured on the surface of the magnetic beads: after the incubation, the mixture was centrifuged briefly, 2. Mu.L of blocked Oligo (dT) beads were added, and the mixture was spun at room temperature for 10min on a spin mixer to capture the immunocomplexes onto the surface of the beads. Discarding the reaction solution after the magnetic beads are completely adsorbed on the magnetic frame, adding 150 mu L of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend the magnetic beads, standing for 1min after short centrifugation, and discarding the supernatant; the supernatant was thoroughly discarded after repeating the "Add rinse buffer-resuspend magnetic beads-rest-discard supernatant" operation 3 times.

5. Ortho ligation reaction: a20. Mu.L ligation system was prepared, which contained 0.1. Mu.L of the splint sequence, 2. Mu. L T4 DNA ligase buffer and 0.4. Mu. L T4 DNA ligase (Thermo), and the ligation system was added to the PCR reaction tube of step 4, and the beads were suspended, and ligation reaction was performed at 37℃for 15 minutes.

6. Eluting: 20. Mu.L of DEPC-H was added to the PCR reaction tube ₂ O, after oscillation transient separation, reacting for 10min at 70 ℃ in a PCR instrument; and (3) after the reaction is finished and the centrifugation is carried out for a short time, placing the mixture on a magnetic rack for timing for 1min, and completely rotating out the clarified elution products after the magnetic beads are completely adsorbed on the magnetic rack.

PCR amplification: a PCR reaction system was configured containing 1. Mu.L of 10 XKod-plus-Neo DNA polymerase buffer, 0.2. Mu.L of Kod-plus-Neo DNA polymerase, 2mM dNTPs, 25mM MgSO ₄ 0.1. Mu.L of upstream primer, 0.1. Mu.L of downstream primer, 5. Mu.L of eluted product; according to 94℃2min,30 cycles (98℃20s,65℃40 s), in a PCR reaction systemAnd (3) performing PCR amplification reaction to obtain a PCR amplification product. The PCR amplified product was purified, its concentration was measured, and second generation sequencing was performed.

Comparative example 1: antigen IL-8 or IL-6 gradient (Single antigen, single antibody pair) was detected singly using conventional PLA methods

In this application, IL-8 or IL-6 is used as the antigen to be analyzed, and the gradient detection of a single antigen, single antibody pair is performed using conventional PLA methods (orthosteric ligation occurs on a solid surface). The relevant steps are summarized as follows:

1. antigen dilution: IL-8 or IL-6 antigen was diluted to the desired concentration using 1 XSSC binding buffer (0.05% Tween 20,0.2% BSA) in a total volume of 50. Mu.L with a maximum antigen concentration of 100pg/mL, 10-fold each dilution yielding a total of 3 antigen concentrations: 100. 10 and 1pg/mL.

2. Antigen-antibody incubation: mu.L of capture sequence R, 0.1. Mu.L of salmon sperm DNA, 0.2. Mu.L of the probe-conjugated antibodies (sequences 2 and 3, or sequences 4 and 5) corresponding to the antigen of example 1, 50. Mu.L of antigen IL-8 or IL-6 were added to 1 XSSC binding buffer gradient, mixed well, and incubated with shaking at 37℃for 1h on a thermostated shaker, so that the antibodies formed immune complexes with the antigen.

3. Immunocomplexes were captured and washed on the surface of the beads: after the incubation, the mixture was centrifuged briefly, 2. Mu.L of blocked Oligo (dT) beads were added, and the mixture was spun at room temperature for 10min on a spin mixer to capture the immunocomplexes onto the surface of the beads. Then, the PCR reaction tube is placed on a magnetic rack for 1min after short centrifugation; discarding the reaction solution after the magnetic beads are completely adsorbed on a magnetic frame, adding 150 mu L of 0.5 XSSC washing buffer solution to resuspend the magnetic beads, standing the magnetic frame, and discarding the supernatant; after repeating the "add wash buffer-rest-discard supernatant" operation 3 times, the supernatant was thoroughly discarded.

4. Ortho ligation reaction: a20. Mu.L ligation system was prepared, which contained 0.1. Mu.L of the splint sequence, 2. Mu. L T4 DNA ligase buffer and 0.4. Mu. L T4 DNA ligase (Thermo), and the ligation system was added to the PCR reaction tube of step 3, and the beads were suspended, and ligation reaction was performed at 37℃for 15 minutes.

5. Cleaning: after the connection reaction, placing the PCR reaction tube on a magnetic rack, and timing for 1min; discarding the reaction solution after the magnetic beads are completely adsorbed on a magnetic frame, adding 150 mu L of 0.5 XSSC washing buffer solution to resuspend the magnetic beads, standing the magnetic frame, and discarding the supernatant; after repeating the "add wash buffer-rest-discard supernatant" operation 3 times, the supernatant was thoroughly discarded.

PCR amplification: a PCR reaction system was configured containing 1. Mu.L of 10 XKod-plus-Neo DNA polymerase buffer, 0.2. Mu.L of Kod-plus-Neo DNA polymerase (Toyobo), 2mM dNTPs, 25mM MgSO ₄ SYBR dye, 0.1. Mu.L upstream primer, 0.1. Mu.L downstream primer, 5. Mu.L eluted product; performing PCR amplification reaction according to 2min at 94 ℃ and 30 cycles (20 s at 98 ℃ and 40s at 65 ℃) under a PCR reaction system to obtain a PCR amplification product; the PCR amplified product was purified, its concentration was measured, and second generation sequencing was performed.

8. Data analysis: qPCR (quantitative polymerase chain reaction) machine starting data, subtracting the Ct value of 0pg/mL from the Ct value of each antigen concentration, taking an absolute value to obtain a-delta Ct value, and expressing the detection capability of the system on the antigen concentration by the-delta Ct value.

Example 3: antigen IL-8 was detected singly using the presently disclosed PLA method and whether the multiple first probes and multiple sample combination sequences interfered with the system (single antigen, single antibody pair)

In this example, IL-8 was used as antigen to be analyzed, gradient detection of single antigen, single antibody pairs was performed using the presently disclosed PLA method (multi-library single preparation), multiple first probes and multiple sample combining sequences were added to simulate sample combining conditions, and whether a system was interfered was detected, and the relevant steps were summarized as follows:

1. First probe, linker scaffold coupled antibody duplex: adding 0.2 mu L of the antibody after coupling the linker scaffold of the IL-8 antigen in example 1 and 0.2 mu L of the first probe (L5) into 48.1 mu L of 1 XSSC binding buffer, and uniformly mixing; incubation is performed on a thermostatted shaker for 10min with shaking at 37℃so that the first probe (L5) forms a double-stranded structure with the adaptor scaffold sequence.

2. Antigen dilution: IL-8 antigen was diluted to the desired concentration using 1 XSSC binding buffer (0.05% Tween 20,0.2% BSA added) to a total volume of 50. Mu.L, antigen concentrations of 100, 10, 1pg/mL in order, and negative of 0pg/mL.

3. Antigen-antibody incubation:

a (without interference factor): 0.2. Mu.L of capture sequence R, 0.1. Mu.L of salmon sperm DNA, 0.2. Mu.L of the first probe of IL-8 antigen obtained in step 1, the double-stranded structure of the linker-scaffold conjugated antibody, 0.2. Mu.L of the second probe conjugated antibody of IL-8 antigen prepared in example 1, 1. Mu.L of sample discrimination Index sequence (SEQ ID NO. 9), and 50. Mu.L of IL-8 antigen were added to 1 XSSC binding buffer, mixed uniformly, and incubated with shaking at 37℃for 1 hour on a thermostatic shaker, so that the antibody forms an immune complex with the antigen.

B (first probe interference): mu.L of capture sequence R, 0.1. Mu.L of salmon sperm DNA, 0.2. Mu.L of the first probe of IL-8 antigen completed in step 1, the double-stranded structure of the linker-scaffold conjugated antibody, 0.2. Mu.L of the second probe conjugated antibody of IL-8 antigen prepared in example 1, 1. Mu.L of sample discrimination Index sequence (SEQ ID NO. 9), 2 kinds of interference first probe (SEQ ID NO. 7, SEQ ID NO. 8) each 1. Mu.L, 50. Mu.L of IL-8 antigen gradient were added to 1 XSSC binding buffer, mixed uniformly, and incubated with shaking at 37℃for 1 hour on a thermostatic shaker, so that the antibody forms an immune complex with the antigen.

4. Immunocomplexes are captured on the surface of the magnetic beads: after the incubation, the mixture was centrifuged briefly, 2. Mu.L of blocked Oligo (dT) beads were added, and the mixture was spun at room temperature for 10min on a spin mixer to capture the immunocomplexes onto the surface of the beads.

5. And (3) cleaning magnetic beads: centrifuging the captured protein low adsorption reaction tube for a short time, standing for 5min at room temperature, placing on a magnetic rack, and timing for 1min; discarding the reaction solution after the magnetic beads are completely adsorbed on the magnetic frame, adding 150 mu L of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend the magnetic beads, standing for 1min after short centrifugation, and discarding the supernatant; the supernatant was thoroughly discarded after repeating the "Add rinse buffer-resuspend magnetic beads-rest-discard supernatant" operation 3 times.

C (sample discrimination sequence interference)

And (3) cleaning magnetic beads: centrifuging the captured protein low adsorption reaction tube for a short time, adding 1 mu L of each sample distinguishing sequence (sequence number 10 and sequence number 11) into the reaction tube, shaking and uniformly mixing, centrifuging for a short time, standing at room temperature for 5min, placing on a magnetic rack, and timing for 1min; discarding the reaction solution after the magnetic beads are completely adsorbed on the magnetic frame, adding 150 mu L of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend the magnetic beads, standing for 1min after short centrifugation, and discarding the supernatant; the supernatant was thoroughly discarded after repeating the "Add rinse buffer-resuspend magnetic beads-rest-discard supernatant" operation 3 times.

6. Ortho ligation reaction: a20. Mu.L ligation system was prepared, which contained 0.1. Mu.L of the splint sequence, 2. Mu. L T4 DNA ligase buffer and 0.4. Mu. L T4 DNA ligase (Thermo), and the ligation system was added to the PCR reaction tube of step 5, and the beads were suspended, and ligation reaction was performed at 37℃for 15 minutes.

7. Eluting: 20. Mu.L of DEPC-H was added to the PCR reaction tube ₂ O, after oscillation transient separation, reacting for 10min at 70 ℃ in a PCR instrument; and (3) after the reaction is finished and the centrifugation is carried out for a short time, placing the mixture on a magnetic rack for timing for 1min, and completely rotating out the clarified elution products after the magnetic beads are completely adsorbed on the magnetic rack.

PCR amplification: a PCR reaction system was configured containing 1. Mu.L of 10 XKod-plus-Neo DNA polymerase buffer, 0.2. Mu.L of Kod-plus-Neo DNA polymerase, 2mM dNTPs, 25mM MgSO ₄ 0.1. Mu.L of upstream primer, 0.1. Mu.L of downstream primer, 5. Mu.L of eluted product; the PCR amplification reaction was performed in a PCR reaction system according to 94℃2min,30 cycles (98℃20s,65℃40 s), to obtain a PCR amplification product. The PCR amplified product was purified, its concentration was measured, and second generation sequencing was performed.

The off-machine data are split into samples according to the Index sequence of the samples, and simultaneously, IL-8 and IL-6 in each sample are quantitatively analyzed according to the barcode sequence coupled with each antibody.

Example 4: antigen IL-8 or IL-6 (Single antigen, single antibody pair) was detected singly using the PLA methods of the present disclosure

In this example, IL-8 or IL-6 was used as the antigen to be analyzed, and the gradient detection of the single antigen, single antibody pair was performed using the PLA method of the present disclosure. The relevant steps are summarized as follows:

1. first probe, linker scaffold coupled antibody duplex: adding 0.2 mu L of the antibody after coupling the linker scaffold corresponding to the antigen in example 1 and 0.2 mu L of the first probe (L5) into 48.1 mu L of 1 XSSC binding buffer solution, and uniformly mixing; incubation is performed on a thermostatted shaker for 10min with shaking at 37℃so that the first probe (L5) forms a double-stranded structure with the adaptor scaffold sequence.

2. Antigen dilution: IL-8 or IL-6 antigen was diluted to the desired concentration using 1 XSSC binding buffer (0.01% Tween 20 added) in a total volume of 50. Mu.L with a maximum antigen concentration of 10000pg/mL, 5-fold each dilution giving a total of 11 antigen concentrations: 10000. 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512 and 0.001024pg/mL.

3. Antigen-antibody incubation: 0.2. Mu.L of capture sequence R, 0.1. Mu.L of salmon sperm DNA, 0.2. Mu.L of the first probe of the corresponding antigen obtained in step 1, the linker-scaffold conjugated antibody double-stranded structure, 0.2. Mu.L of the second probe conjugated antibody of the corresponding antigen prepared in example 1, 1. Mu.L of sample discrimination sequence (each sample using a different sequence), 50. Mu.L of IL-8 or IL-6 antigen gradient were added to 1 XSSC binding buffer, mixed well, and incubated with shaking at 37℃for 1h on a thermostatted shaker, so that the antibodies formed immune complexes with the antigen.

4. The immune complex is captured and washed on the surface of the magnetic bead: after the incubation, the mixture was centrifuged briefly, 2. Mu.L of blocked Oligo (dT) beads were added, and the mixture was spun at room temperature for 10min on a spin mixer to capture the immunocomplexes onto the surface of the beads. Then, the reaction tube with low protein adsorption is put on a magnetic rack for 1min; discarding the reaction solution after the magnetic beads are completely adsorbed on the magnetic frame; adding 150 mu L of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend magnetic beads, standing a magnetic rack, and discarding the supernatant; after resuspension of the beads, 150 μl of 0.5 x ssc wash buffer was added again, and the beads were centrifuged briefly.

A multiple library pool: mixing the products in all the reaction tubes into the same 15mL centrifuge tube, standing on a magnetic rack for 5min after instantaneous centrifugation, and discarding the supernatant; adding 2mL of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend magnetic beads, standing for 1min by a magnetic rack after short centrifugation, and discarding the supernatant; after repeating the 3 "add wash buffer-resuspend beads-rest-discard supernatant" procedure, the supernatant was thoroughly discarded (multiple library pooled preparation).

B multiple library preparation alone: centrifuging the captured protein low adsorption reaction tube for a short time, standing for 5min at room temperature, placing on a magnetic rack, and timing for 1min; discarding the reaction solution after the magnetic beads are completely adsorbed on the magnetic frame, adding 150 mu L of 0.5 XSSC flushing buffer solution, carrying out vortex oscillation to resuspend the magnetic beads, standing for 1min after short centrifugation, and discarding the supernatant; after repeating the 3 "add wash buffer-resuspend beads-rest-discard supernatant" procedure, the supernatant was discarded thoroughly (multiple libraries were prepared separately).

9. Data splitting and analysis: the off-machine data were resolved according to the sample Index sequence, and simultaneously quantitative analysis was performed on IL-8 and IL-6 in each sample according to each antibody-coupled barcode sequence.

Results and discussion

1. Comparison of conventional methods with Single detection results of the methods of the present disclosure (i.e., comparison of the results of comparative example 1 and example 2)

Compared with the conventional PLA scheme, the method disclosed by the disclosure needs to form the probe coupled antibody with a double-chain structure, add sample distinguishing sequences, and have two connecting sites in each sequence, and the steps are obviously different from the conventional PLA scheme. Firstly, it is required to confirm that the biochemical flow can normally occur, taking IL-8 and IL-6 antigens as examples, the antigen concentration is selected to be 0, 1, 10 and 100pg/mL, and the detection condition of the antigen proteins in the two biochemical flows is detected in a qPCR mode.

As shown in fig. 2, the disclosed method detects IL-8 antigen gradients, - Δct is 2.71, 6.65, and 10.76 in order, and conventional PLA is 2.25, 5.72, and 9.08 in order; the disclosed method detects IL-6 antigen gradients, - ΔCt, of 1.86, 4.98 and 8.49 in order, and conventional PLA, of 1.37, 3.73 and 6.60 in order. As can be seen, the method of the present disclosure has a significant gradient trend in the-delta Ct values at different antigen concentration gradients, indicating that the PLA method of the present disclosure can be used for gradient detection of single antigen, single antibody pairs. Meanwhile, compared with the conventional PLA, the detection of the antigen gradient in the scheme disclosed by the disclosure has a larger-delta Ct value, and is presumed to be more beneficial to the detection of low-concentration antigens.

2. The method for detecting the antigen concentration is not influenced by the first probe and the distinguishing sequence of the interference sample Example 3

In the single antigen/single antibody pair measurement (single detection), the interference factor (two first probes, two sample discrimination sequences) equivalent to the experimental reaction is artificially added without interfering the experiment, and when the three antigen/three antibody pair (co-detection) is simulated, the occurrence of crosstalk between libraries can be prevented. Wherein each sample contains only one antigen (IL-8), and the reaction system is added with only one detection antibody pair corresponding to the antigen.

UMI readings represent the amount of antigen in the detected sample, with higher readings indicating higher amounts of antigen detected; UMI reading of the 0 antigen concentration sample represents the detection background value of the detection system; the two phosphorus-added complementary UMI read values of the interference factors represent the crosstalk condition of the single chain in the reaction, and the lower the read value is, the lower the crosstalk risk is; the two Index sequences UMI read values of the interference factor represent the crosstalk condition of the Index sequences when the samples are distinguished, and the lower the read value is, the lower the crosstalk probability is. The interference ratio represents the ratio of UMI read value to detected signal value of the interference factor, and the lower the ratio is, the lower the occurrence rate of the interference condition is.

As shown in fig. 3, taking IL-8 antigen as an example, under the condition of 0pg/mL antigen concentration, the mean value of UMI readings detected by the PLA method of the present disclosure is 101, the mean value of normal signal UMI readings detected by the first probe interference group is 134, and the mean value of interference signal UMI readings detected by the first probe interference group is 1; under the condition of 100pg/mL antigen concentration, the mean value of UMI read values detected by using the PLA method is 45633, the mean value of UMI read values of normal signals detected by the first probe interference group is 55928, and the mean value of UMI read values of interference signals detected by the first probe interference group is 29; the addition of the first probe sequence slightly improves the detection background by 0pg/mL, and the interference ratio is 0.7%; meanwhile, the detection signal of 100pg/mL is improved, and the interference ratio is 0.05%. The first probe sequence is used for being matched with the first antibody connector bracket coupling sequence to form a complex, and antibody codes are distinguished through an index region on the first probe. The addition of multiple first probes does not affect the normally designed signal detection, and the interference ratio is low, thus proving that the scheme has the possibility of jointly detecting multiple antigens.

As shown in fig. 4, taking IL-8 antigen as an example, under the condition of 0pg/mL antigen concentration, the mean value of UMI readings detected by the PLA method of the present disclosure is 529, the mean value of normal signal UMI readings detected by the l sample distinguishing interference group is 824, and the mean value of interference signal UMI readings detected by the sample distinguishing interference group is 4; under the condition of 100pg/mL antigen concentration, the mean value of UMI reading values detected by using the PLA method is 145171, the mean value of UMI reading values of normal signals detected by the sample distinguishing interference group is 152314, and the mean value of UMI reading values of interference signals detected by the sample distinguishing interference group is 523; the addition of the sample distinguishing sequence for interference slightly improves the detection background by 0pg/mL, and the interference proportion is 0.5%; meanwhile, the detection signal of 100pg/mL is improved, and the interference ratio is 0.3%.

Each sample was assigned a specific sample discrimination sequence for sample discrimination after second generation sequencing. The embodiment shows that in the sample merging stage, different sample distinguishing sequences are mixed, normal signal generation is not affected, the interference ratio is low, and visible sample distinguishing sequence signals are not interfered with each other, so that the scheme can be used for merging and detecting alloantigens and multiple samples.

3. The methods of the present disclosure single test multiple library single preparation and multiple library combined preparation comparison (example 4)

As shown in FIG. 5, using the non-pooled sample protocol of the present disclosure, IL-8 had a LoD of 0.1pg/mL and IL-6 had a LoD of 0.21pg/mL; using the sample combining protocol of the present disclosure, IL-8 had a LoD of 0.08pg/mL and IL-6 had a LoD of 0.13pg/mL. The detection LoD of the pooled protocol was only slightly lower than the non-pooled protocol, demonstrating that the pooled protocol did not affect antigen detection sensitivity. Meanwhile, 12 antigen concentrations were performed in this example, each concentration having 3 parallel samples for 36 samples; if a conventional solid-phase PLA scheme or an uncombined scheme of the disclosure is adopted, the incubation of each sample of antigen-antibody needs to be performed for 3-6 times of washing processes, and 108-216 times of washing processes are needed in total; if the sample volume is increased again, it is difficult to operate manually without equipment such as an automated plate washer. According to the sample combining scheme disclosed by the disclosure, the samples are combined after the 'antigen-antibody' incubation, and the combined samples only need to be washed 3-6 times, so that the workload is greatly reduced, and the working efficiency is improved.

All methods described in this disclosure can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples, or exemplary language provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

The present disclosure describes preferred embodiments, including the best mode known to the inventors for carrying out the present disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading this disclosure. These variations may be suitably employed by those skilled in the art, and the present disclosure is intended to cover such variations as may be practiced in a manner different than that specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto. Moreover, unless otherwise indicated herein or clearly contradicted by context, this disclosure is intended to cover any combination of all possible variations of the elements described herein.

Claims

1. A detection method, comprising the following steps in order:

(1) The method comprises the following steps of sequentially carrying out the following steps on more than two samples to be tested: (1-1) mixing a first antibody linked to a first probe, a second antibody linked to a second probe, a sample discrimination sequence and a sample to be tested in a solution to obtain an immune complex; (1-2) contacting the solid surface with the solution obtained in step (1-1) to capture the immune complex to the solid surface; (1-3) washing the solid surface more than once to remove molecules not bound to the solid surface;

(2) Combining solids for more than two samples to be tested;

preferably, the analyte is one or more modified or unmodified proteins.

2. The method of claim 1, wherein the method further comprises, between steps (4) and (5), the steps of:

(4' -1) secondary capturing and cleaning: capturing the immune complex on the solid surface again, cleaning the solid surface for more than one time, and removing molecules which are not bound to the solid surface;

(4' -2) eluting, releasing the immunocomplexes bound on the solid surface into a second elution buffer.

3. The method of claim 1 or 2, wherein in step (5), the detecting comprises multiplex fluorescent quantitative PCR or second generation sequencing.

4. The method of claim 1 or 2, wherein the first antibody, second antibody, first probe and/or second probe has a parking group, the solid surface has a capture group, the parking group is bound to the capture group, thereby capturing the immune complex to the solid surface; preferably, the parking group is selected from one or more of biotin, polydT and polydA, and the capturing group is selected from one or more of streptavidin, avidin, polydT and polydA.

5. The method of claim 1 or 2, wherein the immunocomplex is captured to the solid surface using a capture sequence complementary to the capture region of the first probe and/or the second probe, the capture sequence having a parking group, the solid surface having a capture group, the parking group being bound to the capture group, thereby capturing the immunocomplex to the solid surface; preferably, the parking group is selected from more than one of biotin, polydT, polydA, and the capture group is selected from more than one of streptavidin, polydT, polydA.

6. The method of claim 1 or 2, wherein the ligation system comprises a splint sequence and a ligase; preferably, the ligase is selected from the group consisting of T4 DNA ligase, T7 DNA ligase, splingR ligase, taq ligase.

7. The method of claim 6, wherein the first antibody is coupled with a linker scaffold comprising a region that is compatible with the first probe, a region that is compatible with the sample discrimination sequence, and a 3' terminal modifying group coupled with the first antibody; and/or the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled with the second antibody;

alternatively, the first antibody is coupled with a linker scaffold comprising a region that is interworked with the first probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled with the first antibody; and/or the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 3' terminal modifying group coupled with the second antibody;

8. The method of claim 7, wherein the first and second probes each independently comprise an oligonucleotide strand comprising a second generation sequencing linker region, a barcode region, and a splint sequence complementary region; preferably, the oligonucleotide strand further comprises a UMI region and/or a capture region.

9. The method of claim 8, wherein the first and second probes further have the structure:

the first probe comprises a region that interacts with a linker scaffold, and the second probe comprises a group at the 3 'end that is coupled to an antibody and a phosphorylated group at the 5' end; or,

the second probe comprises a region that interacts with a linker scaffold, and the first probe comprises a group at the 5' end that is coupled to an antibody; or,

the first probe comprises a region that interacts with a linker scaffold, and the second probe comprises a group at the 5' end that is coupled to an antibody; or,

the second probe comprises a region that interacts with a linker scaffold, the first probe comprising a group at the 3 'end that is coupled to an antibody and a phosphorylated group at the 5' end; or,

The first and second probes comprise regions that interact with a linker scaffold;

wherein the group coupled with the antibody is selected from more than one of azide group, amino group and sulfhydryl group; preferably, the sample discrimination sequence comprises a region that interacts with a linker scaffold, a sample barcode region, and a second generation sequencing linker region.

10. The method of claim 8, wherein the proximity ligation reaction product is a nucleic acid reporter comprising a sample discrimination sequence, a UMI region, a barcode region, and/or a splint sequence complementary region of the first and second probes.

11. The method according to claim 1 or 2, wherein in step (1), the solution is a binding buffer selected from one or more of SSC buffer, tris, HEPES, bis-Tris and MOPS, preferably the cation concentration of the binding buffer is 100mM to 1M; preferably, the cation is sodium ion; and/or, in the step (1-3) and the step (4' -1), washing is performed using a washing solution selected from one or more of SSC buffer, tris, HEPES, bis-Tris and MOPS, preferably, the cation concentration of the washing solution is 10mM to 200mM, preferably, the cation is sodium ion.

12. The method according to claim 1 or 2, wherein the first elution buffer is selected from one or more of TE buffer, tris, HEPES, bis-Tris and MOPS, preferably the cation concentration of the first elution buffer is 1mM to 100mM, preferably the cation is sodium ion; and/or the second elution buffer is selected from more than one of DEPC water, RNase/DNase-free water, TE buffer, tris, HEPES, bis-Tris and MOPS.

13. A method according to claim 1 or 2, wherein the solid is selected from magnetic solids such as magnetic beads.

14. A kit for use in the method of any one of claims 1-13, wherein the kit comprises:

(i) A first probe and a first antibody;

(ii) A second probe and a second antibody;

(iii) Sample discrimination sequences;

(xi) Alternative PCR amplification reagents.

15. The kit of claim 14, wherein the first antibody is coupled with a linker scaffold comprising a region that is compatible with the first probe, a region that is compatible with the sample discrimination sequence, and a 3' terminal modifying group coupled with the first antibody; and/or the second antibody is coupled with a linker scaffold comprising a region that is interworked with the second probe, a region that is interworked with the sample discrimination sequence, and a 5' terminal modifying group coupled with the second antibody;

16. The kit of claim 14 or 15, wherein the first and second probes each independently comprise an oligonucleotide strand comprising a second generation sequencing linker region, a barcode region, and a splint sequence complementary region; preferably, the oligonucleotide strand further comprises a UMI region and/or a capture region.

17. The kit of claim 16, wherein the first and second probes further have the structure: