CN-122017245-A - Anti-FAP antibody-magnetic bead conjugate kit and application thereof

Abstract

The invention discloses an anti-FAP antibody-magnetic bead conjugate kit and application thereof, belonging to the field of biological reagents for diagnosis. According to the invention, FAP protein is used as a target to prepare a high-affinity monoclonal antibody, a carboxyl magnetic bead is selected as a solid phase carrier, and EDC/Sulfo-NHS crosslinking agent is used to activate carboxyl on the surface of the magnetic bead, so that the carboxyl forms a stable amide bond with amino on FAP antibody molecules, and a 'carboxyl magnetic bead-amide bond-FAP antibody' covalent coupling structure is constructed. In the structure, the carboxyl magnetic beads provide stable solid phase carriers and high-efficiency magnetic responsiveness, an EDC/Sulfo-NHS crosslinking system ensures firm combination of antibodies and the magnetic beads, an anti-FAP antibody ensures targeting specificity to FAP+EVs, and the three are used for realizing high-efficiency and specific capture and rapid magnetic separation of the FAP+EVs in a synergistic manner, so that the anti-FAP antibody is used for early diagnosis of tumor patients.

Inventors

• HUANG YI
• LIU MING
• LV XIANG
• He Xiamin
• WANG YAOCHENG
• CHEN DONGJIE
• ZHANG BIAO

Assignees

• 福州大学附属省立医院

Dates

Publication Date

20260512

Application Date

20260202

Claims (6)

1. 1. The anti-FAP antibody-magnetic bead conjugate is characterized in that the anti-FAP antibody-magnetic bead conjugate is formed by covalently coupling an anti-FAP antibody and carboxyl magnetic beads through an amide bond.
2. 2. The anti-FAP antibody-magnetic bead conjugate of claim 1, wherein the anti-FAP antibody is covalently coupled to the carboxylic magnetic bead via EDC/Sulfo-NHS dual cross-linker system as a vehicle.
3. 3. A method of preparing an anti-FAP antibody-magnetic bead conjugate according to claim 1, comprising the steps of: (1) The method comprises the steps of (1) activating carboxyl magnetic beads, namely adding 0.1M pH 5.5 MES buffer solution into the carboxyl magnetic beads, placing the carboxyl magnetic beads in an ultrasonic cleaning instrument, carrying out ultrasonic cleaning for 2-6 minutes, carrying out magnetic separation, discarding the liquid, repeatedly cleaning for 2-4 times, then adding 0.1M pH 5.5 MES buffer solution, carrying out ultrasonic mixing to fully suspend the magnetic beads to obtain magnetic bead suspension, weighing EDC and Sulfo-NHS according to the mass ratio of 1:1, respectively dissolving the EDC and the Sulfo-NHS into 8-12 mg/ml mother solution by using the 0.1M pH 5.5 MES buffer solution, immediately adding the mother solution into the magnetic bead suspension after dissolution, carrying out ultrasonic mixing for 0.5-3 minutes, placing the magnetic beads in a constant temperature shaking table at 24-26 ℃ for reacting for 0.8-1.5 hours at the rotating speed of 100-150r/min, and after the reaction is finished, adding the 0.1M pH 5.5 MES buffer solution to carry out ultrasonic cleaning for 2-6 minutes, carrying out magnetic separation, and discarding the liquid to obtain activated carboxyl magnetic beads; (2) The method comprises the steps of antibody coupling, sealing and purifying preservation, namely adding 0.1M pH 9.5 carbonate buffer solution into activated carboxyl magnetic beads, carrying out ultrasonic mixing for 1-4 minutes, adding FAP monoclonal antibody according to the proportion of the magnetic beads to 1mg of the antibody to 20 mug, carrying out gentle inversion and centrifugal tube mixing, carrying out light-shielding reaction for 2-3 hours in a constant temperature shaking table at 24-26 ℃, carrying out rotating speed of 100-150r/min, after the reaction is finished, placing the centrifugal tube on a magnetic frame for magnetic separation, discarding supernatant, adding pH 7.4 PBS buffer solution containing 0.5% BSA, sealing for 1.5-3 hours in a constant temperature shaking table at 36.5-37.5 ℃ after mixing, carrying out rotating speed of 100-150r/min, carrying out magnetic separation liquid waste after sealing, carrying out ultrasonic cleaning for 2-4 times by using pH 7.4 PBS buffer solution containing 0.5% BSA, carrying out magnetic separation liquid waste liquid after each cleaning, and finally carrying out resuspension of the conjugate by using pH 7.4 PBS buffer solution containing 0.5% BSA, and carrying out sealing preservation at 2-8 ℃.
4. 4. A tumor diagnostic kit comprising the anti-FAP antibody-magnetic bead conjugate of claim 1 or 2.
5. 5. Use of an anti-FAP antibody-magnetic bead conjugate according to claim 1 or 2 for capturing and isolating FAP and EVs in a serum sample of a tumor patient.
6. 6. Use of an anti-FAP antibody-magnetic bead conjugate according to claim 1 or 2 for the preparation of a kit for early stage tumor diagnosis.

Description

Anti-FAP antibody-magnetic bead conjugate kit and application thereof Technical Field The invention belongs to the field of biological reagents for diagnosis, in particular to a FAP related disease diagnosis reagent, and particularly relates to an anti-FAP antibody-magnetic bead conjugate kit and application thereof. Background In Tumor Microenvironments (TMEs), cancer-associated fibroblasts (CAF) are core stromal cells that regulate tumor proliferation, metastasis, and immune escape. CAF is formed by activation of Normal Fibroblasts (NF) by tumor signals, whose functional specificity stems from the differential expression of surface markers. Fibroblast Activation Protein (FAP) is a specific target of CAF, and is taken as a transmembrane serine protease, and is highly expressed on the surface of CAF of epithelial tumors such as lung cancer, gastric cancer and the like, but hardly expressed in normal NF. The exosomes secreted by the CAF carry FAP and other active substances, are not only TME communication carriers, but also have tumor diagnosis value. Extracellular Vesicles (EVs) are a type of membranous vesicles released by cells and play an important role in cell communication, disease occurrence and the like. Biomolecules (such as proteins, RNA, and DNA) carried by EVs can be used as biomarkers for early detection of cancer. These specific molecular markers can be identified by blood detection, not only with high sensitivity and specificity, but also for monitoring therapeutic response and disease progression. EVs can be classified into exosomes, microvesicles, apoptotic bodies, etc. according to the secretion mode, the microvesicles are directly excreted outside due to the excretion effect of the cytoplasmic membrane, and the apoptotic bodies are shriveled or split vesicles due to apoptosis of cells. Unlike other EVs, exosomes occur from the inward recession of the parent endocytosis to form Multivesicles (MVBs), via early and late endosomes, MVBs can dynamically communicate with other organelles or cellular compartments (e.g., trans-golgi networks, endoplasmic reticulum, mitochondria, phagosome, RNA particles, micronuclei, etc.). Different types of cargo such as proteins, RNA, DNA or lipids are classified into MVBs. After MVBs maturation, they can be degraded by fusion with lysosomes, or by fusion with cell membranes to release internal vesicles (EVs), so-called exosomes. It is possible that exosomes carry some of the membrane proteins of the parental cells, and therefore we reasonably speculate that the expression of membrane protein FAP is also present in bilayer lipid membranes of EVs such as CAF releasing exosomes. The detection of serum EVs provides a potential noninvasive biomarker and a molecular treatment target for liquid biopsy of tumors, but the detection of the serum EVs is key to accurately diagnosing, monitoring and prognosis evaluation of tumor patients by capturing EVs derived from tumors from various cells in the body and detecting protein markers related to tumor progress. In recent years, the role of CAF derived EVs in tumor progression has attracted attention. Research shows that CAF can release EVs, and plays an important role in the aspects of tumor growth, metastasis, treatment resistance and the like by carrying protein, miRNA and other components of extracellular matrix, regulating metabolism, inhibiting immune response, promoting angiogenesis and maintaining tumor cell stem property. Based on the above research, we reasonably consider that the serum CAF source exosomes and other EVs are enriched so as to detect the EVs protein markers related to tumor progression, thereby being beneficial to providing new serological markers and potential molecular therapeutic targets for tumor prevention and treatment. The prior art only uses FAP as a detection marker, such as patent CN 115166242A only uses anti-FAP reagent to detect exosome FAP, and no targeting separation is involved. In other technologies, the single antibody method is easy to destroy exosomes or high in cost, and the target recognition of the aptamer method is single and is easy to be interfered, so that the requirements cannot be met. The core problem is that CAF derived EVs overlap with other EVs in physical properties and lack a highly efficient coupling system for CAF specific markers. Therefore, the development of FAP-targeted, high-purity and high-stability EVs separation tools is a key for breaking through the bottleneck of tumor liquid biopsy. Disclosure of Invention In view of the shortcomings of the prior art, a first object of the present invention is to provide an anti-FAP antibody-magnetic bead conjugate based on FAP specific targeting, so as to solve the problem that the prior art cannot efficiently and specifically capture CAF-derived EVs from a complex serum sample, thereby realizing high purity enrichment of fap+evs, and simultaneously guaranteeing structural integrity of EVs to meet subsequent detection r