CN120442620B - Oligonucleotide blockers for protein detection systems and methods for reducing background in ortho-reaction-based ultrasensitive protein detection systems - Google Patents
Oligonucleotide blockers for protein detection systems and methods for reducing background in ortho-reaction-based ultrasensitive protein detection systemsInfo
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Abstract
本发明公开了用于蛋白检测体系的寡核苷酸阻断剂及降低基于邻位反应的超灵敏蛋白检测体系背景的方法，该寡核苷酸阻断剂包括至少一对寡核苷酸序列，每条寡核苷酸序列均包括核心阻断序列以及分别处于该核心阻断序列5’端和3’端的支撑序列，仅该核心阻断序列与探针中的延伸位点部分或完全互补。其中，核心阻断序列能占据无靶标的游离抗体‑探针偶联物的延伸位点，使无靶标的游离抗体‑探针偶联物不能相互结合延伸，从而降低PEA超灵敏蛋白检测背景；而支撑序列不仅能防止核心阻断序列自身形成二级结构，而且能防止空间扭曲或刚性拉力导致的核酸配对效率下降，稳定寡核苷酸阻断剂的阻断效果。
This invention discloses an oligonucleotide blocker for protein detection systems and a method for reducing the background of ultrasensitive protein detection systems based on ortho-reactions. The oligonucleotide blocker comprises at least one pair of oligonucleotide sequences, each oligonucleotide sequence including a core blocking sequence and support sequences located at the 5' and 3' ends of the core blocking sequence, respectively. Only the core blocking sequence is partially or completely complementary to the extension site in the probe. The core blocking sequence occupies the extension site of the untargeted free antibody-probe conjugate, preventing the untargeted free antibody-probe conjugate from binding and extending, thereby reducing the background of PEA ultrasensitive protein detection. The support sequences not only prevent the core blocking sequence from forming secondary structures itself, but also prevent the decrease in nucleic acid pairing efficiency caused by spatial distortion or rigid tension, stabilizing the blocking effect of the oligonucleotide blocker.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an oligonucleotide blocker for a protein detection system and a method for reducing the background of an ultrasensitive protein detection system based on ortho-position reaction.
Background
The protein detection technology has important significance in biomedical research and clinical diagnosis, and especially in early screening, disease course monitoring and curative effect evaluation of serious diseases, the detection sensitivity directly determines the application depth and accuracy of the protein detection technology. The traditional detection technology such as ELISA (enzyme-linked immunosorbent assay), chemiluminescence and the like has certain quantitative capability, but the lower detection limit is usually at the pM level, and the requirement of accurately detecting low-abundance protein markers at the fg/mL level and even the ag/mL level in blood plasma cannot be met. Along with the continuous promotion of accurate medical treatment and early intervention, the development of protein detection means with higher sensitivity, smaller sample requirement and faster response capability has become a common requirement of clinic and scientific research.
In recent years, the rising ultrasensitive detection technology, such as a single-molecule immunization method (Simoa) of Quanterix company, has the detection lower limit reaching fM or even aM level, has 100-1000 times higher detection sensitivity than the traditional technology, and can detect and analyze the protein markers with ultralow abundance in blood plasma. However, the method based on Simoa technology platform involves a multi-step cleaning process, requires a matched precise chip and expensive instruments, so that the detection cost is high, and the method is difficult to be applied to large-scale clinical diagnosis.
Besides Simoa technology platforms, rapid protein detection technologies based on proximity reaction, such as ortho ligation reaction (Proximity Ligation Assay, PLA) and ortho extension reaction (ProximityExtension Assay, PEA), are developed in recent years, ultra-sensitive plasma protein detection can be realized, advantages of no cleaning, compatibility with qPCR and the like are achieved, and the method is suitable for rapid detection and automation platform integration. PLA and PEA are both homogeneous systems, relying on diabodies to recognize targets to trigger nucleic acid signal amplification, but have substantial differences in their enzymatic reaction mechanisms. PLA relies on DNA ligase to achieve nucleic acid ligation, and reaction efficiency is often inhibited by interfering substances in complex samples such as plasma. The PEA replaces ligase with DNA polymerase, so that the reaction efficiency is improved, and the PEA is widely applicable to liquid phase environments such as plasma and the like. However, there are background noise problems caused by nonspecific binding of free probes in both PLA and PEA.
In order to reduce the background signal of the ortho-reaction system, two strategies exist at present, namely, a solid-phase PLA cleaning mode is adopted to reduce the background, and a non-specific protein sealing mode is adopted to reduce the background. For example, patent CN117431300a proposes a method for improving detection sensitivity by blocking the hybridization site between the probe and the capture probe, and assisting the washing operation. However, the method introduces multiple times of cleaning, and breaks down the essential advantages of no-cleaning and rapid detection of PLA. Patent CN103154266B discloses a protein-nucleic acid coupled blocking agent that prevents false positive signal amplification by binding to non-target proteins in a sample. Although these strategies alleviate the background noise problem to some extent, the former relies on solid phase systems and physical washing, and the latter lacks site specificity, and none is suitable for background control due to non-specific extension between free antibody-probe conjugates.
Disclosure of Invention
In order to solve the background noise problem caused by nonspecific extension between free antibody-probe conjugates in an ortho-extension reaction (PEA) system, the invention provides a blocker designed based on short-chain oligonucleotides, which can specifically occupy a pair of probe extension sites and realize effective background control in a liquid phase system which does not depend on washing.
The technical scheme of the invention is as follows:
an oligonucleotide blocker for a protein detection system comprising at least a pair of oligonucleotide sequences, each comprising a core blocking sequence and a support sequence at the 5 'and 3' ends of the core blocking sequence, respectively, only the core blocking sequence being partially or fully complementary to an extension site in a probe.
The invention designs an oligonucleotide blocker structure in a universal oligonucleotide single-chain form by analyzing the nucleic acid sequence of an extension site in an antibody-probe conjugate used by an ortho-extension method. The single strand of the oligonucleotide blocker has a core blocking sequence which is partially or completely complementary to the probe and a supporting sequence which is completely not complementary to the probe, wherein the core blocking sequence can occupy an extension site of a free antibody-probe conjugate without a target, so that the free antibody-probe conjugate without the target cannot be combined and extended mutually, thereby reducing the background of a PEA system (the affinity between a pair of antibody-probe conjugates which capture the target is stronger than that between the oligonucleotide blocker and the antibody-probe conjugate, and the oligonucleotide blocker can be replaced out in a competitive way so as to expose the extension site), and the supporting sequence can effectively prevent a secondary structure from being formed in a core region and relieve the reduction of pairing efficiency caused by spatial conformational stress, thereby enhancing the structural stability and functional persistence of the blocker.
In the oligonucleotide blocker of the present invention, the core blocking sequence is designed according to the nucleotide sequence of the probe used, and the nucleotide sequence of the probe is universal in the PEA system and is not changed according to the target analytes. Therefore, it is considered that the specific nucleotide sequence of the core blocking sequence of the present invention is not limited, and it is only necessary to design it based on the selected probe sequence under the conditions defined in the present invention.
Preferably, in the oligonucleotide blocking agent, the number of nucleotides of the core blocking sequence is 10 or less.
In the two oligonucleotide sequences belonging to the same pair, the lengths of the two core blocking sequences can be the same or different, and the length is preferably controlled below 10 nucleotides, and experiments show that when the length of the core blocking sequence is too large, the blocking effect is reduced, and the synthesis cost is increased.
Preferably, in the oligonucleotide blocker, at least 4 continuous complementary bases are arranged between the core blocking sequence and the extension site of the probe, namely 4-10 continuous complementary bases are arranged between the core blocking sequence and the extension site of the probe.
However, in the two oligonucleotide sequences of the same pair, the two core blocking sequences may be partially or completely complementary to the extension site of the probe independently, and the invention is not particularly limited thereto. In a preferred embodiment, the core blocking sequence of at least one oligonucleotide sequence is fully complementary to the extension site in the probe to provide a better blocking effect.
Preferably, in the oligonucleotide blocker, at least 4 consecutive complementary bases are present between the two core blocker sequences in the two oligonucleotide sequences belonging to the same pair, and the number of consecutive complementary bases between the two core blocker sequences is smaller than the number of consecutive complementary bases between the core blocker sequence and the extension site of the probe, and more preferably, 4 to 6 consecutive complementary bases are present between the two core blocker sequences.
The number of complementary bases between the core blocking sequences is typically less than the number of complementary bases between the core blocking sequences and the probes, which on the one hand reduces the likelihood of complementarity between the oligonucleotide blockers and on the other hand ensures preferential binding of the core blocking sequences to the extension sites of the probes.
Preferably, in the oligonucleotide blocker described above, the support sequence has at least 3 to 6 consecutive bases T.
The invention also provides a method for reducing the background of an ultrasensitive protein detection system based on ortho-position reaction, which comprises the following steps:
(1) Incubating the oligonucleotide blocker with an antibody-probe conjugate and a sample to be analyzed;
(2) Adding an extension solution, and carrying out extension reaction after uniformly mixing;
(3) And continuously adding the qPCR system, uniformly mixing, performing qPCR reaction, and analyzing the qPCR detection result.
It can be seen that the oligonucleotide blocker of the invention is very simple to use, can effectively reduce the background signal of an ortho-extension assay system without depending on specific enzymes or additional washing steps, thereby improving the sensitivity and specificity of detection, providing a new solution for high-throughput and ultrasensitive protein detection, and being suitable for analyzing a wide range of samples.
Preferably, in step (1) of the above method, the concentration ratio of the oligonucleotide blocker to the antibody-probe conjugate is (100-2000): 1, and the working concentration of the antibody-probe conjugate is 100-500pM;
in step (1), 10-20 min were incubated at 37 ℃.
In step (1) of the above method, the antibody-probe conjugate may be prepared by any method existing or unknown, and the present invention is not limited to this, and as an example of a specific embodiment, may be prepared by the following method:
(a) Activating the antibody by adopting a TCO-PEG4-NHS reagent to obtain an activated antibody;
(b) Activating the amino modified probe by adopting a Tz-PEG4-NHS reagent to obtain an activated probe;
(c) And coupling the activated antibody and the activated probe to obtain the antibody-probe conjugate. Preferably, in step (2) of the above application, the composition of the extension solution is 4. Mu.L of 10 Xbuffer, 4. Mu.L of 100. Mu.M equimolar mixture of four deoxynucleotide triphosphates, 2. Mu.L of 8000U/mL Bst elongase, 26. Mu.L of deionized water;
The extension reaction flow is that the extension is carried out for 20min at 37 ℃ and the inactivation is carried out for 20min at 80 ℃;
in step (3), the qPCR system consisted of 0.4. Mu.L of each qPCR detection primer, 0.2. Mu.L of 6-carboxyfluorescein, 10. Mu.L of commercial qPCR mixture containing fluorescent dye and polymerase, and 5. Mu.L of deionized water.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, through analyzing the nucleic acid sequence of an extension site in an antibody-probe conjugate used in an ortho-extension method, a novel oligonucleotide blocker in a universal oligonucleotide single-chain form is designed, wherein the single chain of the oligonucleotide blocker is provided with a core blocking sequence which is partially or completely complementary with a probe and a supporting sequence which is not completely complementary with the probe, wherein the core blocking sequence can occupy the extension site of a free antibody-probe conjugate without a target, so that the free antibody-probe conjugate without the target cannot be combined and extended mutually, the nonspecific background of a PEA system is reduced (the affinity between a pair of antibody-probe conjugates capturing the target is stronger than that between the oligonucleotide blocker and the antibody-probe conjugate, so that the oligonucleotide blocker can be replaced by the competitively, the extension site is exposed), and the supporting sequence can not only prevent the core blocking sequence from forming a secondary structure, but also prevent the nucleic acid pairing efficiency from being reduced due to space distortion or rigid pulling force, and stabilize the blocking effect of the oligonucleotide blocker.
(2) The oligonucleotide blocker of the invention is very simple and convenient to use, can effectively reduce the background signal of an ortho-extension method analysis system under the condition of not depending on specific enzymes or additional cleaning steps, thereby improving the sensitivity and the specificity of detection, providing a new solution for high-flux and ultrasensitive protein detection, and being suitable for analyzing a wide range of samples.
(3) When the oligonucleotide blocker of the present invention is used for detecting proteins by the ortho extension method, the signal of the target protein can be enhanced by more than 2.5 times.
Drawings
FIG. 1 is a schematic diagram of the working principle of the oligonucleotide blocker of the present invention;
wherein, cycles represents cycle number, fluorescence represents Fluorescence signal, negative represents Negative, threshold represents critical value, positive represents Positive;
FIG. 2 shows the blocking effect of different concentrations of the oligonucleotide blocking agent of the invention when assayed for IL-6 by ortho extension;
wherein Concentration represents the IL-6 concentration, the same as below;
FIG. 3 shows the blocking effect of the oligonucleotide blockers of the invention having different nucleotide sequences when assayed for IL-6 by ortho extension;
FIG. 4 shows the blocking effect of the oligonucleotide blocking agent of the invention in the ortho-extension assay of IL-6 at various antibody-probe conjugate concentrations;
FIG. 5 shows the blocking effect of the oligonucleotide blocking agent of the invention when assayed for PSA in the orthostretching method;
FIG. 6 shows the blocking effect of the oligonucleotide blocking agent of the invention when assayed for IL-17 by ortho extension.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and examples.
Examples 1-7 design of oligonucleotide blockers
In an embodiment of the invention, an oligonucleotide blocker is provided, comprising at least one pair of oligonucleotide sequences, each of which consists of a core blocker sequence and a support sequence at the 5 'and 3' ends of the core blocker sequence, respectively, wherein only the core blocker sequence is partially or completely complementary to the extension site of the probe and the support sequence is not completely complementary to the extension site of the probe.
Wherein the number of nucleotides of the core blocking sequence is less than or equal to 10, and at least 4 continuous complementary bases are arranged between each core blocking sequence and an extension site in the probe.
In the two oligonucleotide sequences of the same pair, there are 4-6 consecutive complementary bases between the two core blocking sequences.
The support sequence consists of 3-6 consecutive bases T.
To describe the specific properties of the oligonucleotide blockers, the examples of the present invention, following the principles outlined above, design several pairs of oligonucleotide blockers on the basis of the following probes:
Probe-1:5 '-GTGAGGCCAGCGTCTTTTATATTAGGCCCTGGTATAGCAGACTGAAA-3' (SEQ ID No. 1);
probe-2:5 '-CGCAATGTCGCACATGATTCTCTGACGAACCGCTTTGCCTGATTTCAGTCT-3' (SEQ ID No. 2).
The nucleotide sequences of each pair of oligonucleotide blockers are shown in Table 1.
Table 1 nucleotide sequences of the oligonucleotide blockers of each pair
| F(5’→ 3’) | R(5’→ 3’) | |
| Oligonucleotide blocker 1 | TTTACACAGTACTTT(SEQ ID No.3) | TTTAGACTGAAATTT(SEQ ID No.4) |
| Oligonucleotide blocker 2 | TTTAAACAGTCTTTT(SEQ ID No.5) | TTTTTAGACTGAAATTTTT(SEQ ID No.6) |
| Oligonucleotide blocker 3 | TTTAATCAGTCTTTT(SEQ ID No.7) | TTTTTAGACTGAAATTTTTT(SEQ ID No.8) |
| Oligonucleotide blocker 4 | TTTTTTCAGTCTTTT(SEQ ID No.9) | TTTTTTAGACTGAAATTTTTT(SEQ ID No.10) |
| Oligonucleotide blocker 5 | TTTTTTCAGTCTGTTT(SEQ ID No.11) | TTTAGACTGAAATTT(SEQ ID No.12) |
| Oligonucleotide blocker 6 | TTTTTTCAGTCTGCTTTT(SEQ ID No.13) | TTTAGACTGAAATTT(SEQ ID No.14) |
| Oligonucleotide blocker 7 | TTTTTTCAGTCTGCTATTTT(SEQ ID No.15) | TTTAGACTGAAATTT(EQ ID No.16) |
Note that the core blocking sequence is underlined and bolded, and the italics are bases complementary to the probe extension site.
The working principle of the oligonucleotide blocker of this example is shown in FIG. 1. It can be seen that the core blocking sequence of the oligonucleotide blocker first binds to the probe extension site on the antibody-probe conjugate, and when the sample to be analyzed is added, the antibody portion of the antibody-probe conjugate binds to the target, and at this time, the affinity between the pair of antibody-probe conjugates capturing the target is stronger than the affinity between the oligonucleotide blocker and the antibody-probe conjugate, so that the oligonucleotide blocker is competitively displaced to expose the extension site, and the extension site is extended after the two are bound, and the extension site of the free antibody-probe conjugate not capturing the target is still occupied by the oligonucleotide blocker, so that the free antibody-probe conjugate without the target cannot be bound to each other for extension, thereby reducing the background noise of the PEA system.
Examples 8-11 oligonucleotide blocker 1 was used to analyze blocking effects when IL-6
A method for reducing the background of ultrasensitive protein detection based on proximity reaction in this embodiment, the method comprising the steps of:
(1) Incubating the oligonucleotide blocker 1 with an antibody-probe conjugate and a sample to be analyzed;
Wherein, the antibody-probe conjugate can be prepared by the following method:
(a) Activating the antibody by adopting a TCO-PEG4-NHS reagent to obtain an activated antibody;
Specifically, firstly centrifuging a desalting column meeting the quality requirement of a target antibody to remove a storage buffer solution, then centrifuging and replacing the desalting column for 3 times by using 0.1M NaHCO 3 +PBS, adding 10 mug of antibody (OriGeneD 624,624, D623) into a replaced filter column, centrifuging, adding a coupling agent TCO-PEG4-NHS with the amount of 1/10 antibody substance into filtrate, and performing light-proof reaction at room temperature for 25 min to obtain a reaction solution A;
Then, centrifuging a desalting column meeting the antibody quality requirement to remove the storage buffer solution, and centrifuging and replacing the storage buffer solution for three times by using PBS buffer solution;
(b) Activating the amino modified probe by adopting a Tz-PEG4-NHS reagent to obtain an activated probe;
specifically, firstly centrifuging a desalting column meeting the quality requirement of a probe to remove a storage buffer solution, and then centrifuging and replacing the desalting column with a borate buffer solution (0.1 mol/L PH=8.5) for three times, wherein 10 mu L of DNA modified by NH 2 is taken and put in a filter column, and after centrifuging, tz-PEG4-NHS with the amount of 1/20 of substances is added, and the reaction liquid B is obtained by carrying out light-proof reaction at room temperature for 25 min;
Then, centrifuging a desalting column meeting the probe quality requirement to remove the storage buffer solution, and centrifuging and replacing the storage buffer solution for three times by using PBS buffer solution;
(c) Coupling the activated antibody and the activated probe to obtain the antibody-probe conjugate;
specifically, uniformly mixing the desalted activated antibody and activated probe according to the ratio of 1:1, and carrying out light-shielding reaction at room temperature for 45min to obtain an antibody-probe conjugate;
In the real step, the PEA Buffer is used for diluting the antibody-probe conjugate to the concentration of 500pM for standby, then the oligonucleotide blocker and the antibody-probe conjugate are respectively mixed uniformly according to the proportion of 100:1, 300:1, 500:1, 800:1, 1000:1, 1500:1 and 2000:1, then a sample to be analyzed (IL-6 concentration is 1000, 200, 40, 8, 1.6, 0.32, 0.064 and 0 pg/mL) is added, and the mixture is incubated for 15 min at 37 ℃;
(2) Adding an extension solution, and carrying out extension reaction after uniformly mixing;
The composition of the extension solution in this example was 4. Mu.L of 10 Xbuffer, 4. Mu L A/T/C/GTP, 2. Mu.L of 8000U/mL Bst elongase, 26. Mu.L of deionized water;
After the extension solution is uniformly mixed with the incubation liquid, the mixture is put into a PCR instrument for extension reaction, wherein the extension reaction process is that the extension is carried out for 20min at 37 ℃ and the inactivation is carried out for 20min at 80 ℃;
(3) Continuously adding a qPCR system, uniformly mixing, performing qPCR reaction, and analyzing a qPCR detection result;
In this example, the qPCR system consisted of 0.4. Mu.L of qPCR detection primer, 0.2. Mu.L of FAM, 10. Mu.L of Mix Buffer, 5. Mu.L of deionized water;
Wherein the qPCR detection primer comprises:
An upstream primer GTGAGGCCAGCGTCTTTTATATTA (SEQ ID No. 17);
A downstream primer CAATGTCGCACATGATTCT (SEQ ID No. 18);
qPCR reaction procedure is shown in table 2:
table 2 qPCR reaction procedure
After the qPCR reaction was completed, the CT values were compared in different cases to evaluate the inhibition of PEA non-specific background signal by the oligonucleotide blocker, the results are shown in table and fig. 2.
TABLE 3 analysis of blocking Effect of oligonucleotide blocker 1 at different concentrations in IL-6 by ortho extension
Note that fold refers to the concentration of oligonucleotide blocker relative to antibody-probe conjugate, ΔCT represents the quantitative detection cycle difference between the concentration of 1000 pg/mL and the blank background of 0 pg/mL, representing the signal intensity of the detection.
As can be seen from the results of Table 3 and FIG. 2, the ΔCT values without the addition of the oligonucleotide blocker were 8.51, whereas the ΔCT values were 9.64, 9.97, 10.15, 9.86, 10.29, 9.78, 9.58, respectively, when the oligonucleotide blocker was added 100-fold, 300-fold, 600-fold, 800-fold, 1000-fold, 1500-fold and 2000-fold, i.e., the background CT of the ultrasensitive protein detection was reduced due to the blocking effect of the oligonucleotide blocker, such that the signal of the target analyte IL-6 was enhanced 2.19-fold, 2.75-fold, 3.11-fold, 2.55-fold, 3.43-fold, 2.41-fold, 2.10-fold, respectively.
Also, as can be seen from the results of Table 2 and FIG. 2, the appropriate oligonucleotide blocker fold is advantageous for further increasing the blocking efficiency, with a more pronounced signal enhancement of the target analyte at 300-1000 fold.
Examples 12-17 different oligonucleotide blockers were used to analyze blocking effects when IL-6
The protein detection method based on the ortho-extension method is basically the same as that of the embodiment 5, and is different in that the adopted oligonucleotide blocking agents comprise an oligonucleotide blocking agent 2, an oligonucleotide blocking agent 3, an oligonucleotide blocking agent 4, an oligonucleotide blocking agent 5, an oligonucleotide blocking agent 6 and an oligonucleotide blocking agent 7 in sequence.
The case of oligonucleotide blocker 1 and no oligonucleotide blocker was set again at the same time as a comparison.
The concentration ratio of each oligonucleotide blocker to antibody-probe conjugate was 1000:1.
The blocking effect of each oligonucleotide blocker is shown in table 4 and fig. 3.
TABLE 4 blocking effect of different oligonucleotide blockers in the ortho extension assay for IL-6
As can be seen from Table 4 and FIG. 3, the ΔCT values without the addition of oligonucleotide blocker were 8.77, and the ΔCT values with the addition of oligonucleotide blocker 1, oligonucleotide blocker 2, oligonucleotide blocker 3 and oligonucleotide blocker 4 were 10.08, 10.19, 10.22, 10.25, respectively, with a decrease in the background CT of ultrasensitive protein detection due to the blocking effect of the oligonucleotide blocker, resulting in an increase in the signal of target analyte IL-6 by a factor of 4.60, 4.96, 5.06, 5.17, respectively.
However, when oligonucleotide blocker 5, oligonucleotide blocker 6, and oligonucleotide blocker 7 were added, the ΔCT values were 9.83, 9.20, and 9.04 in this order, and the signal of target analyte IL-6 was increased 2.08-fold, 1.34-fold, and 1.21-fold, respectively.
Examples 18-20 Effect of different ratios of oligonucleotide blocking agent to antibody-probe conjugate on blocking Effect
The method for detecting the protein based on the ortho-extension method is basically the same as that of the embodiment 5, except that the concentration of the antibody-probe conjugate is sequentially 100pM, 200 pM and 400pM, and the ratio of the oligonucleotide blocker 1 to the antibody-probe conjugate is 1000:1.
The case where the concentration of the antibody-probe conjugate was again set to 500 pM was also used as a comparison.
The blocking results are shown in table 5 and fig. 4.
TABLE 5 Effect of probe concentration on blocking effect
As can be seen from Table 5 and FIG. 4, the signal intensity of IL-6 was 1.09-1.93-fold different at different concentrations of the antibody-probe conjugate, wherein the blocking effect was optimal when the concentration of the antibody-probe conjugate was 100 pM.
Example 21 blocking Effect of oligonucleotide blocking agent 1 when used to analyze PSA
The method for detecting protein based on the ortho-extension method is basically the same as that of example 5, except that the target analyte is PSA, the concentration of the antibody-probe conjugate is 200pM, and the concentration ratio of the oligonucleotide blocker 1 to the antibody-probe conjugate is 1000:1.
The analysis results are shown in Table 6 and FIG. 5.
TABLE 6 oligonucleotide blocker 1 blocking effect in PSA in ortho extension assay
As can be seen from Table 6 and FIG. 5, the signal of PSA was enhanced by a factor of 2.08 after the addition of oligonucleotide blocker 1 compared to the absence of oligonucleotide blocker.
Example 22 oligonucleotide blocker 1 was used to analyze blocking effects when IL-17
The method for detecting a protein based on the ortho-extension method of this example is basically the same as that of example 5, except that the target analyte is IL-17, the concentration of the antibody-probe conjugate is 100pM, and the concentration ratio of the oligonucleotide blocker 1 to the antibody-probe conjugate is 1000:1.
The analysis results are shown in Table 7 and FIG. 6.
TABLE 7 oligonucleotide blocker 1 analysis of blocking effect in IL-17 in ortho extension
As can be seen from Table 7 and FIG. 6, the IL-17 signal was increased by a factor of 2.68 with the addition of oligonucleotide blocker 1 compared to the absence of oligonucleotide blocker.
Claims (6)
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| WO2024062116A1 (en) * | 2022-09-23 | 2024-03-28 | Navinci Diagnostics Ab | Padlock blocking oligonucleotide |
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| EP1994180A4 (en) * | 2006-02-24 | 2009-11-25 | Callida Genomics Inc | High throughput genome sequencing on dna arrays |
| GB201107863D0 (en) * | 2011-05-11 | 2011-06-22 | Olink Ab | Method and product |
| BR112021019640A2 (en) * | 2019-12-19 | 2022-06-21 | Illumina Inc | High-throughput single cell libraries and methods of preparation and use |
| IL300973A (en) * | 2020-09-02 | 2023-04-01 | Meso Scale Technologies Llc | Kits for detecting one or more target analytes in a sample and methods of making and using the same |
| CN113528611A (en) * | 2021-06-01 | 2021-10-22 | 上海交通大学 | Protein detection kit and detection method |
| CN116790715A (en) * | 2023-01-18 | 2023-09-22 | 良渚实验室 | A high-throughput ultrasensitive centrifugal digital protein detection method |
| WO2025072857A1 (en) * | 2023-09-28 | 2025-04-03 | Guardant Health, Inc. | Methods for multiplexed protein detection |
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| WO2024062116A1 (en) * | 2022-09-23 | 2024-03-28 | Navinci Diagnostics Ab | Padlock blocking oligonucleotide |
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