Systems and methods for disease detection

Abstract

The present invention provides methods and kits for the analysis of extracellular vesicle-derived biomarkers obtained from dried bodily fluid samples, including but not limited to blood spots extracted on or into an absorbent surface, for the analysis of extracellular vesicles for disease diagnosis, staging, monitoring, and therapeutic drug selection and efficacy. The present invention also provides methods and apparatus for sample collection for home or field use. The apparatus and methods of the present invention are useful for the collection of any bodily fluid sample.

Description

Technical Field

[0001] Technical Field The present invention relates to devices and methods for sample collection and testing.

Background Art

[0002] Background Appropriate sample collection is critical for the delivery of high-quality healthcare. Many conventional diagnostic tests are performed on venous blood samples. However, these conventional approaches have drawbacks. Some people may be reluctant or unable to provide blood at a collection center. For example, many individuals in rural areas and elderly people often have difficulty traveling to clinical laboratories. In addition, anxiety about venipuncture contributes to reduced compliance. Therefore, clinic visits may contribute to disengagement from necessary medical care. Accordingly, there is a need for techniques that improve access to diagnostic tests.

Summary of the Invention

Means for Solving the Problems

[0003] Summary The present invention provides a method and apparatus for sample collection for use at home or in the field. The apparatus and method of the present invention are useful for collecting any bodily fluid sample. According to the present invention, the bodily fluid is placed on an absorbent matrix and dried, and then the matrix (or apparatus containing the matrix) is shipped to a laboratory for analysis. In one embodiment, the clinical laboratory isolates extracellular vesicles from the dried spot on the collection apparatus for analysis. Extracellular vesicles provide specific disease biomarkers associated with tissues, for example, tumor cells. Tumor cells release extracellular vesicles into circulation, so the presence of cancer can be detected by probing bodily fluids such as blood. According to the present invention, extracellular vesicles can be preserved intact in a dried blood spot or other similar sample and subsequently recovered from there. A feature of the present invention is that the dried bodily fluid sample (e.g., blood) can be sent to a reference laboratory by mail or courier service. Thus, the present invention provides a detection kit with instructions for collecting one or more samples on a collection card which is then sent to a laboratory. In a preferred embodiment, the collection card comprises a matrix on which body fluids are deposited and which promotes the retention and elution of extracellular vesicles.

[0004] Another feature of the present invention is that samples can be collected multiple times over a specified period to increase the likelihood of obtaining representative samples (for example, after fasting or at a specific time during the day). Since sample collection can be performed at home, certain geographical, personal, or financial obstacles are minimized.

[0005] In certain embodiments, the present invention provides an analytical method comprising obtaining a sample collection device having a dried bodily fluid sample (e.g., blood) on its surface, extracting at least one extracellular vesicle from the dried bodily fluid sample, detecting at least one biomarker associated with the extracellular vesicle, and determining the patient's health status based on the biomarker. In a preferred embodiment, the health status is determined using the simultaneous appearance of at least three biomarkers on the extracellular vesicle. The three biomarkers are preferably selected based on their simultaneous appearance in cells indicating the medical condition to be diagnosed.

[0006] The bodily fluids used in the method of the present invention may be obtained, for example, by fingertip puncture and by transporting the fluid onto an absorbent matrix (e.g., cellulose). The collection device comprises a matrix for receiving the sample. The sample is dried on the matrix within the device. The matrix may be pre-treated with sugars, additives, surfactants, antimicrobial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, anticoagulants, and / or drying agents. The method and apparatus of the present invention may also include a mailer and instructions for returning the obtained sample to the reference laboratory.

[0007] In some embodiments, extracellular vesicles are extracted from a dried sample spot by elution into an aqueous mixture. The extracellular vesicles are analyzed by detecting the presence of disease-related biomarkers. In preferred embodiments, the biomarkers are located on the surface of the vesicles. In certain assays, disease diagnosis is achieved by analyzing multiple simultaneously present biomarkers on the vesicles, for example, using a proximity ligation assay. For example, the assay may involve conjugating two oligonucleotides to each of two biomarkers, ligating the two oligonucleotides to a single nucleic acid construct, and detecting the nucleic acid construct. Preferably, the proximity ligation assay uses two antibody-DNA conjugates. The DNA portions of the two antibody-DNA conjugates can be ligated together and detected by a reaction such as quantitative PCR.

[0008] A particular aspect of the present invention provides a sample collection device. A preferred sample collection device comprises a substrate (such as cardstock or plastic), at least one absorbent matrix on the surface of the substrate, a lancet, and a sterilizer (e.g., ethyl alcohol). The device may include surface markings to indicate to the user where to place the body fluid sample, and at least one capillary structure for drawing the body fluid sample into the absorbent matrix. The absorbent matrix may be pre-treated with sugars, additives, surfactants, antimicrobial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, anticoagulants, or desiccants. The absorbent matrix may be filter paper, cellulose, paper, or some other fibrous material. Preferably, the absorbent matrix is ​​soluble in aqueous solution. In some embodiments, the absorbent matrix has an open internal structure to facilitate fluid absorption by capillary action, and an internal space sized to receive released extracellular vesicles and retain them intact. The sterilizer may be ethyl alcohol (e.g., allowing the user to wipe and disinfect their fingers to provide an uncontaminated sample). The apparatus may be shipped to the testing facility via postal service (for example, with shipping information pre-printed). In a preferred embodiment, the absorbable matrix contains reagents for elution and isolation of extracellular vesicles.

[0009] In the multi-spot embodiment, the surface of the support has at least (i) a first sample collection location including an absorbent matrix and (ii) a second sample collection location including a second absorbent matrix. The device may be accompanied by a package including a lancet, a return envelope, and instructions for collecting multiple samples.

[0010] Any type of bodily fluid, such as blood, sputum, or urine, can be used according to the present invention. In a preferred embodiment, the bodily fluid is blood. The blood is obtained via fingertip puncture using a capillary tube or lancet. In one embodiment, the blood is placed on an absorbent matrix and air-dried. The absorbent matrix may be filter paper, cellulose, paper, or any similar absorbent material.

[0011] Aspects of the present invention include providing a user with a home collection device for collecting bodily fluid samples. A preferred collection device comprises an absorbent material, a sterilizer, a capillary or lancet for blood collection, and packaging for returning the collection device to the laboratory for further analysis. The user collects blood spots on an absorbent matrix, air-dries the spots, and returns them to the laboratory, where the dried blood spots are analyzed for the presence of disease-specific biomarkers.

[0012] In a preferred embodiment, extracellular vesicles are eluted from a dried blood spot for disease detection. In a preferred embodiment, the extracellular vesicles are isolated from the dried blood spot using size exclusion. The isolated extracellular vesicles are assayed for the presence of disease-related biomarkers. The present invention utilizes any molecular biology technique for identifying, characterizing, analyzing, and / or quantifying disease-related biomarkers on the surface of extracellular vesicles. Such techniques include, but are not limited to, PCR, antibody-DNA conjugates, and detection probes. In one non-limiting embodiment, an extracellular vesicle having co-localization of at least three or more surface biomarkers is considered a target extracellular vesicle. The target extracellular vesicle comes into contact with a capture agent directed to the extracellular vesicle-related surface biomarkers. In some embodiments, such a capture agent may include a binding moiety directed to the extracellular vesicle-related surface, which may optionally be conjugated to a solid support. An exemplary capture agent for an extracellular vesicle-related membrane-binding polypeptide may include a solid support (e.g., magnetic beads) and a binding moiety directed to the extracellular vesicle-related membrane-binding polypeptide (e.g., an antibody-active substance). The captured extracellular vesicles are incubated with a pair of antibody-DNA conjugates having double-stranded DNA containing complementary single-stranded overhangs. When the antibody-DNA conjugates bind to two biomarkers co-occurring on the same extracellular vesicle, the single-stranded overhang portions of the antibody-DNA conjugates hybridize with each other. Such a combination, including the extracellular vesicle and the pair of detection probes, is then maintained under conditions that allow the pair of detection probes to bind to their respective targets on the extracellular vesicle, such that the oligonucleotide domains are close enough to anneal and form a double-stranded complex. Such a double-stranded complex can be detected by contacting the double-stranded complex with a nucleic acid ligase to create a ligated template, and then detecting the ligated template. In some embodiments, the ligated template can be detected using quantitative PCR. The presence of such a ligated template indicates the presence of an extracellular vesicle that is positive for the disease-targeted biomarker signature.

[0013] The present invention is useful for detecting all kinds of diseases, but is particularly applicable to the diagnosis and screening of cancer. The present invention also envisions a method for analyzing disease progression, including severity, staging, and recurrence (including minimal residual disease). For example, changes in certain biomarkers (increases or decreases in quantity, structural changes, sequence changes) are known to be associated with certain diseases. Therefore, the method of the present invention tracks biomarkers in extracellular vesicles for changes indicating disease severity or staging, for example. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the sample collection device.

[0015] [Figure 2] Figure 2 shows extracellular vesicles in an aqueous suspension in a tube.

[0016] [Figure 3] Figure 3 illustrates the steps of a proximity ligation assay.

[0017] [Figure 4] Figure 4 shows the steps of the analytical method of the present invention. [Modes for carrying out the invention]

[0018] Detailed explanation The present invention generally relates to methods and apparatus for disease sample collection and detection. In particular, the present invention provides methods and apparatus for detecting diseases by analyzing extracellular vesicles in dried bodily fluids for the presence of disease-specific biomarkers.

[0019] Figure 1 shows a sample collection device 101 for collecting a body fluid sample 103. An exemplary sample collection device comprises a support 109 having an absorbent matrix 113 on its surface. The device 101 may also include a reagent 133 and, optionally, additional reagents 134, which are pre-loaded, for example, on the support 109 or on enclosed packaging or package 105. For example, reagent 133 may be a sterilizing agent (e.g., ethyl alcohol). The additional reagent 134 may be an analgesic or local anesthetic such as lidocaine. The device may include surface markings to indicate to the user where to place the body fluid sample and at least one capillary structure for drawing the body fluid sample into the absorbent matrix. The absorbent matrix may be pre-treated with sugars, additives, surfactants, antimicrobial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, anticoagulants, or desiccants. The absorbent matrix 113 may be filter paper, cellulose, paper, or any other fibrous material. In some embodiments, the absorbent matrix 113 is a material soluble in aqueous solution. Preferably, the absorbent matrix 113 has an open internal structure that promotes blood absorption by capillary action and an internal space sized to receive extracellular vesicles 201 and retain them intact.

[0020] When using the device, the person places the bodily fluid sample 201 on the indicated spot and allows the sample to dry. Instructions to that effect may be printed on the packaging paper 105. The user may then ship the device 101 to the testing facility via postal service or courier.

[0021] The support 109 may be provided as a cardstock piece or a plastic piece. In some embodiments, the surface of the support has at least (i) a first sample collection location including an absorbent matrix and (ii) a second sample collection location including a second absorbent matrix. The device 101 may be provided as part of a package including a lancet, a return envelope, and instructions for collecting multiple samples.

[0022] Any suitable fluid sample can be collected on the device 101. In a preferred embodiment, the device 101 is used to collect a bodily fluid sample. The bodily fluid may preferably be saliva, urine, sweat, or blood. In a particularly preferred embodiment, the bodily fluid is blood. A blood spot is obtained via fingertip puncture using a capillary or a lancet. The blood spot is collected on an absorbent matrix and allowed to dry. The absorbent matrix 113 may be cellulose, paper (e.g., filter paper), cotton gauze, fleece, polymeric paper, or other absorbent materials. The absorbent matrix may be pre-treated with one or any combination of sugars, additives, surfactants, antibacterial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, anticoagulants, and desiccants. The absorbent matrix 113 having the dried blood spot 103 can be mailed by the user to a laboratory or testing facility for further processing and analysis.

[0023] The present invention enables easy at-home sample collection and shipment for users without the need to travel to a testing facility to provide a sample. The present invention is also useful for predicting whether a person is susceptible to a disease, or for detecting progression, or for predicting onset and / or recurrence. The present invention can be utilized to detect all types of diseases. In a preferred embodiment, the disease is cancer. For example, a person diagnosed with cancer or undergoing cancer treatment may collect and dry a blood spot 103 on the absorbent matrix 113 at home, package it, and ship the collection device 101 to a laboratory to detect or stage the cancer. Depending on the disease stage, a physician can select a treatment plan for the person. Similarly, a person who has recovered from cancer can provide a dried blood spot to predict cancer recurrence.

[0024] When the absorbent matrix with the dried blood spot deposited thereon is mailed to a testing facility, the sample can be prepared for further analysis without the need for additional processing of the sample. In one embodiment, the absorbent matrix is soluble in an aqueous solution used to elute extracellular vesicles 201 from the dried blood spot. One simple approach for extracting EV 201 from the spot 103 is to remove a disc of the absorbent matrix 113 (having the EV 201 therein) from the device 101, for example, by punching a hole through the support 109 using a punch. The disc of the absorbent matrix 113 can be dropped into water in a suitable tube or container. If necessary, a size selection step (e.g., via a chromatograph or the like) can be performed to specifically purify the solution for EV 110. In one embodiment, extracellular vesicles are eluted and isolated from the dried blood spot using size exclusion chromatography. As noted above, the matrix can be constructed to extract and retain extracellular vesicles.

[0025] Figure 2 shows EV 110 in solution 205 within tube 207. Once the extracellular vesicles 110 are eluted into the aqueous solution 205, the extracellular vesicles are analyzed for biomarkers. In a preferred embodiment, the extracellular vesicles are analyzed for the co-occurrence of 3 or more biomarkers. In a preferred embodiment, the co-occurrence of biomarkers is analyzed using magnetic beads 231 decorated with antibody 235 against the first of the three biomarkers. The capture assay includes the step of contacting the target extracellular vesicles with at least one capture agent. In a preferred embodiment, the capture agent can be magnetic beads 231 having capture antibody 235. The capture agent binds to at least one or more biomarkers present on the surface of the extracellular vesicles. Once captured, an assay is performed to detect one or more disease-specific biomarkers on the surface of the extracellular vesicles.

[0026] Figure 3 shows the steps of a proximity ligation assay that yields a positive result only if, or only if, the second and third biomarkers of the three biomarkers are present on the extracellular vesicle 110. The proximity ligation assay includes conjugating two antibodies, each linked to oligonucleotides 307 and 308, to the second and third biomarkers, ligating the two oligonucleotides to nucleic acid construct 341, and detecting the nucleic acid construct 341. As shown, the proximity ligation assay uses two antibody-DNA conjugates, but other ligands or reagents may be used. The DNA portions of the two antibody-DNA conjugates, oligonucleotides 307 and 308, are ligated together. The nucleic acid construct 341 can be detected by a suitable reaction such as quantitative PCR (qPCR).

[0027] Figure 4 shows the steps of analytical method 401 of the present invention. Method 401 includes obtaining a sample collection device having a dried bodily fluid sample (e.g., blood) on its surface, extracting at least one extracellular vesicle from the dried bodily fluid sample, detecting the simultaneous appearance of at least three biomarkers on the extracellular vesicle, and reporting the presence of a medical condition based on the detected simultaneous appearance of the three biomarkers. The fluid may be obtained via fingertip puncture and / or using capillary tubes. The collection device may have an absorbent matrix on its surface, in which the bodily fluid sample is dried. The absorbent matrix 113 may be pretreated with sugars, additives, surfactants, antimicrobial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, anticoagulants, desiccants, or any combination thereof. Method 401 may include, prior to the acquisition step, placing the bodily fluid sample on the surface and sending the sample collection device to a person along with instructions for air-drying the bodily fluid sample. Method 401 may include receiving the sample collection device 101 by mail.

[0028] Preferably, the extraction step is carried out by eluting extracellular vesicles into an aqueous mixture. In some embodiments, the analytical step includes a binding assay for detecting three biomarkers. For example, a first biomarker may be captured by an antibody attached to magnetic beads to purify EVs from solution. Second and third biomarkers may be detected by proximity ligation assays. Preferably, the three biomarkers are selected based on their co-occurrence in cells with a medical condition such as cancer.

[0029] The apparatus and methods of the present invention may use any combination (or “panel” or “biomarker signature”) for any particular disease, for example, for lung cancer, as described in International Publication No. 2022 / 011197, incorporated herein by reference. For the analysis of breast cancer, the methods and apparatus of the present invention may use the markers disclosed in International Publication No. 2023 / 004078, incorporated herein by reference. For the analysis of prostate cancer, the methods and apparatus of the present invention may use the markers disclosed in International Publication No. 2023 / 004082, incorporated herein by reference. For the analysis of pancreatic cancer, the methods and apparatus of the present invention may use the markers disclosed in International Publication No. 2023 / 004080, incorporated herein by reference. For the analysis of colorectal cancer, the methods and apparatus of the present invention may use the markers disclosed in International Publication No. 2023 / 004077, incorporated herein by reference.

[0030] Furthermore, the present invention provides a test kit for home use. The test kit allows users to obtain a fluid spot at home and then mail the kit to a testing facility for further analysis. The kit of the present invention comprises a sterilizer for sterilizing the skin, a capillary or lancet for obtaining a blood spot by fingertip puncture, an absorbent matrix for collecting and drying the blood spot, and a shipping label that allows the user to mail the kit to a testing facility. [Examples]

[0031] Example 1 A collection kit comprising an absorbent surface, disinfectant wipes, and a lancet is presented to the user. The user applies disinfectant and then obtains a blood droplet using the lancet, depositing the blood spot onto the absorbent surface, in this case, filter paper. The filter paper is pre-treated with a substance that preserves extracellular vesicles. The blood spot is air-dried by exposing the filter paper to air for a certain period of time according to the included instructions. The dried blood spot deposited on the filter paper is mailed to the laboratory. Upon receiving the dried blood spot on the filter paper, the filter paper is placed in a tube containing phosphate-buffered solution (PBS) for 1 hour. The eluate is passed through a size exclusion chromatography (SEC) column to isolate target extracellular vesicles based on their size. The extracellular vesicles are analyzed for the presence of lung adenocarcinoma biomarkers. If the sTn, MUC1, and CEACAM6 biomarkers are detected together on the EV by the method of this disclosure, the presence of lung adenocarcinoma is reported.

[0032] Example 2 Whole blood was collected via fingertip puncture and deposited on Whatman #2 and Whatman #3 filter paper. The samples were either eluted as provided or spiked with COV413A extracellular vesicles (obtained from cancer cell lines). The blood spots were dried and eluted from the filter paper. Two combinations were used. One focused on three tetraspanins (CD63, CD81, CD9) ubiquitously expressed on extracellular vesicles. The second combination was an ovarian cancer combination (MUC1, BST2+FOLR1) that gave a strong signal only when the blood was spiked with COV413A extracellular vesicles. The signal significantly decreased upon addition of a washing agent, suggesting that the majority of the signal originated from the extracellular vesicles. One of the samples was passed through a size exclusion chromatography (SEC) column after elution from the filter paper. This was done to ensure that no cell fragments that could produce a misleading signal were present in the sample. Even from the SEC-treated sample, a strong signal was present. Data from cleaning agents and samples suggest that extracellular vesicles form the majority of the signals obtained from dried blood spots.

[0033] The presented data were analyzed, and ΔCt was obtained by subtracting the Ct of each group from the blank sample (Whatman #2 paper containing PBS). A larger ΔCt indicates a stronger signal. The results show that the signal in blood without the washing agent was stronger than the signal in blood with the washing agent. Finally, COV413A EV was added to the paper in PBS. The signal was weaker than expected. The difference may be due to the fact that extracellular vesicles in blood are protected by serum proteins and are better preserved than extracellular vesicles added in buffer.

Claims

1. A method for evaluating the disease state: A step of obtaining a sample collection device having a dried bodily fluid sample on its surface; The steps include: extracting at least one extracellular vesicle from the dried body fluid sample; A step of detecting the presence of a biomarker on the extracellular vesicle; A step of evaluating the disease state based on the presence of the biomarker. Methods that include...

2. The method according to claim 1, further comprising the step of sending the sampling device to an individual for sample collection.

3. The method according to claim 1, wherein the detection step includes detecting a plurality of extracellular vesicle-related biomarkers.

4. The method according to claim 3, wherein the plurality of extracellular vesicle-related biomarkers include three biomarkers present on a single extracellular vesicle.

5. The method according to claim 1, wherein the bodily fluid is blood.

6. The method according to claim 1, wherein the surface includes an absorbent matrix.

7. The method according to claim 6, wherein the matrix is ​​pretreated with sugar, additives, surfactants, antibacterial agents, antifungal agents, proteinase inhibitors, DNase inhibitors, or desiccants.

8. The method according to claim 1, wherein the extraction step is performed by eluting extracellular vesicles into an aqueous mixture.

9. The method according to claim 1, wherein the analytical step includes a binding assay for detecting the biomarker.

10. The method according to claim 4, wherein a first biomarker is bound to magnetic beads by an antibody, and a second biomarker and a third biomarker are detected by proximity ligation assay.

11. The method according to claim 10, wherein the proximity ligation assay comprises binding two oligonucleotides to the second biomarker and the third biomarker, ligating the two oligonucleotides to a nucleic acid construct, and detecting the nucleic acid construct.

12. The method according to claim 10, wherein the magnetic beads are used to hold the extracellular vesicles during the washing process.

13. The method according to claim 11, wherein the proximity ligation assay uses two antibody-DNA conjugates.

14. The method according to claim 10, wherein the DNA portions of the two antibody-DNA conjugates are ligated together.

15. The method according to claim 4, wherein the three biomarkers are selected based on the simultaneous appearance of the three biomarkers in cells having a medical condition.

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