Antibody Complexes Research Articles

IL-2/anti-IL-2 antibody complexes augment immune responses to therapeutic cancer vaccines

One driver of the high failure rates of clinical trials for therapeutic cancer vaccines is likely the inability to sufficiently engage conventional dendritic cells (cDCs), the antigen-presenting cell (APC) subset that is specialized in priming antitumor T cells. Here, we demonstrate that, relative to vaccination with an injectable mesoporous silica rod (MPS) vaccine alone (Vax), combining MPS vaccines with CD122-biased IL-2/anti-IL-2 antibody complexes (IL-2cx) drives ~3-fold expansion of cDCs at the vaccination sites, vaccine-draining lymph nodes, and spleens of treated mice. Furthermore, relative to Vax alone, Vax+IL-2cx led to a ~3-fold increase in the numbers of CD8 + T cells and ~15-fold increase in the numbers of NK cells at the vaccination site. Notably, with both the model protein antigen OVA as well as various peptide neoantigens, Vax+IL-2cx induced ~5 to 30-fold greater numbers of circulating antigen-specific CD8 + T cells relative to Vax alone. We further demonstrate that Vax+IL-2cx leads to significantly improved efficacy in the MC38 colon carcinoma model relative to either monotherapy alone, driving complete regressions in 50% of mice in a cDC-dependent manner. Relative to vaccine alone, Vax+IL-2cx led to comparable numbers of CD8 + T cells, but markedly greater numbers of NK cells and activated cDCs in the B16F10 melanoma tumor microenvironment post-therapy. Taken together, these findings suggest that the administration of factors that engage both the cDC-CD8 + T cell and cDC-NK cell axes can boost the potency of therapeutic cancer vaccines.

Dextran-Encapsulated Nanoparticles and Super-Nanoparticle Assemblies: Preparation from Quantum Dots, Fluorescent Polymers, and Magnetic Nanoparticles for Application to Cellular Immunolabeling.

Nanoparticles (NPs) continue to be developed as labels for bioanalysis and imaging due to their small size and, in many cases, emergent properties such as photoluminescence (PL) and superparamagnetism. Some applications stand to benefit from amplification of the advantageous properties of a NP, but this amplification is not a simple matter of scaling for size-dependent properties. One promising approach to amplification is, therefore, to assemble many copies of a NP into a larger but still nanoscale and colloidal entity. Here, we use multiple types of hydrophobic nanocrystal to show that amphiphilic dextran is a versatile material for the preparation and surface functionalization of such super-NP assemblies: CdSe/CdS/ZnS quantum dots (QDs), InP/ZnS QDs, and Si QDs; iron oxide magnetic NPs (MNPs); composites of QDs and MNPs; and composites of QDs and MNPs with fluorene-based and phenylenevinylene-based conjugated polymers. The amphiphilic dextran was also useful for the preparation of conjugated polymer NPs (CPNs) without the inclusion of inorganic nanocrystals. The prepared super-NPs and CPNs were characterized, physically and photophysically, at both the ensemble and the single-particle levels. Per colloidal entity, the super-QDs were orders of magnitude brighter than the individual QDs. This enhancement enabled assemblies of nominally more benign InP/ZnS and Si QDs to be competitive alternative materials to CdSe/CdS/ZnS QDs, which are normally much brighter when compared as individual nanocrystals. The dextran functionalization imparted low nonspecific binding and enabled the use of tetrameric antibody complexes (TACs) for simple and selective immunolabeling of cells with all of the prepared super-NP, CPN, and composite materials. Labeling with the super-QDs provided significantly enhanced PL signals, the super-MNPs enabled magnetic pull-down of cells, and both capabilities were concurrently available with composite assemblies. Overall, this study demonstrates that the preparatory method and functional benefits of amphiphilic dextran extend to a range of hydrophobic materials and combinations thereof. There is strong potential for assembling a diverse set of property-amplified designer labels that are ready-made for in vitro applications in bioanalysis and imaging.

The Effects of the oxLDL/β2GPI/anti-β2GPI Complex on Macrophage Autophagy and its Mechanism.

Previous research has established that the oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex can stimulate macrophages to secrete molecules associated with atherosclerosis (AS), such as monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), and tumor necrosis factor-α (TNF-α). This complex also enhances the uptake of oxLDL, thereby accelerating foam cell formation through the Toll-like receptor-4/nuclear factor kappa B (TLR4/NF-κB) pathway. Given the critical role of macrophage autophagy in the instability of vulnerable atherosclerotic plaques, it is imperative to investigate whether the oxLDL/β2GPI/anti-β2GPI complex influences macrophage autophagy in AS. This study aims to elucidate the effects and underlying mechanisms of the oxLDL/β2GPI/anti-β2GPI complex on macrophage autophagy in AS. Experiments were conducted using murine macrophage RAW264.7 cells and the human monocytic cell line THP-1. Western blot analysis was employed to determine the expressions of autophagy-associated markers and signaling pathway proteins. Autophagosomes were detected through mRFP-GFP-LC3 adenoviral transfection and transmission electron microscopy (TEM). Treatment of macrophages with the oxLDL/β2GPI/anti-β2GPI complex resulted in decreased expressions of Beclin1 and LC3 proteins, alongside an upregulation of SQSTM1/P62 protein expression. Additionally, there was a reduction in the number of autophagosomes and autolysosomes. An increase in the phosphorylation levels of phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) was also observed. Notably, the expressions of autophagy-associated markers were partially restored when the TLR4/NF-κB and PI3K/AKT/mTOR pathways were inhibited by their respective inhibitors. Our findings indicate that the oxLDL/β2GPI/anti-β2GPI complex inhibits macrophage autophagy in AS via the TLR4/NF-κB and PI3K/AKT/mTOR signaling pathways.

Identification of peripheral nerve functional fascicles in Sprague-Dawley rats by the carbon quantum dot-Annexin V antibody complex

To explore a method to identify the sensory and motor fascicles of the peripheral nerve to achieve accurate peripheral nerve functional fascicle suture. The peripheral nerve Sunderland V injury model, muscle branch of the femoral nerve and saphenous nerve were established in the bilateral femoral nerves of Sprague-Dawley (SD) rats. The specific samples were grouped as follows: the main trunk of the femoral nerve was exposed bilaterally and cut with microscopic scissors in the main trunk of the femoral nerve to prepare a model of Sunderland V injury in the mixed fascicle of peripheral nerves; the muscle branch of the femoral nerve was exposed bilaterally and cut in the middle section of the muscle branch of the femoral nerve to prepare a model of Sunderland V injury in the motor fascicle of peripheral nerves; the saphenous nerve was exposed bilaterally and cut at 1 cm below the patella to prepare a model of Sunderland V injury to the sensory fascicle of the peripheral nerves. A carbon quantum dot (CD)-annexin V antibody complex was prepared and applied to the distal and proximal nerve stumps of the peripheral nerve Sunderland V injury model groups of SD rats. Under the excitation light source of a 380 nm uv lamp, fluorescence color development was observed under a fluorescence microscope after 5, 10, 15, and 20 min. At 5 min, sections of the bilateral femoral nerve trunk, muscular branches of the femoral nerve, and Sunderland V lesion of the saphenous nerve in SD rats were only dark in color under the microscope, and there was no difference in fluorescence. The intensity of the staining increased significantly for 10–20 min. The sensory fascicles and saphenous nerves of the femoral nerve trunk showed blue fluorescence under the CD-Annexin V antibody complex staining, while the motor fascicles and muscle branches of the femoral nerve trunk showed no fluorescence. Fluorescence intensity gradually decreased after 20 min of staining. There was no significant difference in the staining intensity at 5, 10, 15, and 20 min in each group. Our results suggest that the CD-Annexin antibody complex can be used to identify functional fascicles of peripheral nerves in SD rats.

In Silico Modeling of Myelin Oligodendrocyte Glycoprotein Disulfide Bond Reduction by Phosphine-Borane Complexes

Background: Neurodegenerative diseases can cause vision loss by damaging retinal ganglion cells in the optic nerve. Novel phosphine-borane compounds (PBs) can protect these cells from oxidative stress via the reduction of disulfide bonds. However, the specific targets of these compounds are unknown. Proteomic evidence suggests that myelin oligodendrocyte glycoprotein (MOG) is a potential target. MOG is of significant interest due to its role in anti-MOG optic neuritis syndrome. Methods: We used in silico modeling to explore the structural consequences of cleaving the extracellular domain MOG disulfide bond, both in isolation and in complex with anti-MOG antibodies. The potential binding of PBs to this bond was examined using molecular docking. Results: Cleaving the disulfide bond of MOG altered the structure of MOG dimers and reduced their energetic favorability by 46.13 kcal/mol. The energy profiles of anti-MOG antibody complexes were less favorable when the disulfide bond of MOG was reduced in the monomeric state by 55.21 kcal/mol, but the reverse was true in the dimeric state. PBs exhibited reducing capabilities with the MOG extracellular disulfide bond, with this best-scoring compound binding with an energy of −28.54 kcal/mol to the MOG monomer and −24.97 kcal/mol to the MOG dimer. Conclusions: These findings suggest that PBs can affect the structure of MOG dimers and the formation of antibody complexes by reducing the MOG disulfide bond. Structural changes in MOG could have implications for neurodegenerative diseases and anti-MOG syndrome.

Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail

Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD.

HRP-anti-HRP sea star primitive antibody complex: first modelization in 3d. invertebrate ig sites in 3d

HRP-anti-HRP sea star primitive antibody complex: first modelization in 3d. invertebrate ig sites in 3d

TCR3d 2.0: expanding the T cell receptor structure database with new structures, toolsand interactions.

Recognition of antigens by T cell receptors (TCRs) is a key component of adaptive immunity. Understanding the structures of these TCR interactions provides major insights into immune protection and diseases, and enables design of therapeutics, vaccinesand predictive modeling algorithms. Previously, we released TCR3d, a database and resource for structures of TCRs and their recognition. Due to the growth of available structures and categories of complexes, the content of TCR3d has expanded substantially in the past 5 years. This expansion includes new tables dedicated to TCR mimic antibody complex structures, TCR-CD3 complexesand annotated Class I and II peptide-MHC complexes. Additionally, tools are available for users to calculate docking geometries for input TCR and TCR mimic complex structures. The core tables of TCR-peptide-MHC complexes have grown by 50%, and include binding affinity data for experimentally determined structures. These major content and feature updates enhance TCR3d as a resource for immunology, therapeuticsand structural biology research, and enable advanced approaches for predictive TCR modeling and design. TCR3d is available at: https://tcr3d.ibbr.umd.edu.

A chorismate mutase-targeted, core-shell nanoassembly-activated SERS immunoassay platform for rapid non-invasive detection of Acanthamoeba infection

Contact lens care and early diagnosis of Acanthamoeba keratitis (AK) are very important to prevent progression to blindness due to AK, which develops when Acanthamoeba attaches to contact lens-damaged corneas. Therefore, we propose a novel, non-invasive, immuno-surface-enhanced Raman scattering (SERS) sensing platform for rapid and accurate detection of Acanthamoeba infection in the tears and contact lens solutions of humans. This optic analysis method was based on the proven biological performance of chorismate mutase (CM)-specific monoclonal and polyclonal antibodies on trophozoite and cyst forms of Acanthamoeba castellanii, and its conditioned media. SERS-based, ultra-low concentration detection was achieved by the anisotropic fanblade-shaped core-shell nanoassembly (Ag@AuFNP) embedded with 4-fluorobenzenethiol Raman reporter. The immuno-SERS platform combining Ag@AuFNP and CM-specific antibody complexes was evaluated in vitro and in vivo. The non-invasive SERS-activated biosensing platform indicates strong feasibility for AK detection in human tears and contact lens solutions.

MXene-Polydopamine-antiCEACAM1 Antibody Complex as a Strategy for Targeted Ablation of Melanoma.

Photothermal therapy (PTT) is a method for eradicating tumor tissues through the use of photothermal materials and photosensitizing agents that absorb light energy from laser sources and convert it into heat, which selectively targets and destroys cancer cells while sparing healthy tissue. MXenes have been intensively investigated as photosensitizing agents for PTT. However, achieving the selectivity of MXenes to the tumor cells remains a challenge. Specific antibodies (Ab) against tumor antigens can achieve homing of the photosensitizing agents toward tumor cells, but their immobilization on MXene received little attention. Here, we offer a strategy for the selective ablation of melanoma cells using MXene-polydopamine-antiCEACAM1 Ab complexes. We coated Ti3C2Tx MXene with polydopamine (PDA), a natural compound that attaches Ab to the MXene surface, followed by conjugation with an anti-CEACAM1 Ab. Our experiments confirm the biocompatibility of the Ti3C2Tx-PDA and Ti3C2Tx-PDA-antiCEACAM1 Ab complexes across various cell types. We also established a protocol for the selective ablation of CEACAM1-positive melanoma cells using near-infrared irradiation. The obtained complexes exhibit high selectivity and efficiency in targeting and eliminating CEACAM1-positive melanoma cells while sparing CEACAM1-negative cells. These results demonstrate the potential of MXene-PDA-Ab complexes for cancer therapy. They underline the critical role of targeted therapies in oncology, offering a promising avenue for the precise and safe treatment of melanoma and possibly other cancers characterized by specific biomarkers. Future research will aim to refine these complexes for clinical use, paving the way for new strategies for cancer treatment.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Synthesis of chitosan polyethylene glycol antibody complex for delivery of Imatinib in lung cancer cell lines.

Lung cancer is known as the most common cancer. Although the Ramucirumab antibody is a second-line treatment for lung cancer, the high interstitial fluid pressure limits the antibody's performance. In this way, Imatinib is a chemotherapeutic drug to reduce the interstitial fluid pressure. Up to now, unfortunately, both Ramucirumab and imatinib have not been reported in one nanosystem for cancer therapy. To fulfill this shortcoming, this paper aims to design a chitosan nanocarrier that loads imatinib and attaches to Ramucirumab for selective bonding to A549. Therefore, this paper aims to develop a polymeric nanosystem for non-small cell lung cancer (NSCLC) treatment. In first, the chitosan polyethylene glycol nanoparticle is synthesized, loaded with imatinib, and then targeted using Ramucirumab. Afterwards, the CS-PEG-Ab-Im by FTIR, TEM, DLS, zeta potential, and TGA techniques are characterized. The size of CS-PEG-Ab-Im was 25-30 nm, its surface charge was 13.1 mV, and the shape of CS-PEG-Ab-Im was nearly spherical and cylindrical. The therapeutic potential of CS-PEG-Ab-Im was assessed using the A549 cell line. According to the obtained results, the cell viability was 48% after 48 h of treatment of A549 cells using the IC50 concentration of CS-PEG-Ab-Im (100 nanomolar). Moreover, the apoptosis and cell cycle arrest percentages were increased by 3 and 6 times, respectively, as compared to free imatinib. Furthermore, the release rate of imatinib from CS-PEG-Ab-Im in an acidic medium was 17% during 1 h, indicating five times the imatinib release in the natural medium. Eventually, the result of flow cytometry indicates the more apoptotic effect of nanosystem to free imatinib and CS-PEG-Ab. Besides, cell arresting result exhibits the CS-PEG-Ab-Im and causes cell arrested at G1 by %8.17. Thus, it can be concluded that CS-PEG-Ab-Im can be an ideal nanosystem in NSCLC treatment.

Unsupervised evolution of protein and antibody complexes with a structure-informed language model.

Large language models trained on sequence information alone can learn high-level principles of protein design. However, beyond sequence, the three-dimensional structures of proteins determine their specific function, activity, and evolvability. Here, we show that a general protein language model augmented with protein structure backbone coordinates can guide evolution for diverse proteins without the need to model individual functional tasks. We also demonstrate that ESM-IF1, which was only trained on single-chain structures, can be extended to engineer protein complexes. Using this approach, we screened about 30 variants of two therapeutic clinical antibodies used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We achieved up to 25-fold improvement in neutralization and 37-fold improvement in affinity against antibody-escaped viral variants of concern BQ.1.1 and XBB.1.5, respectively. These findings highlight the advantage of integrating structural information to identify efficient protein evolution trajectories without requiring any task-specific training data.

A novel anti-CD47 protein antibody and toll-like receptor agonist complex detects tumor surface CD-47 changes in early stage lung cancer by in vivo imaging

CD47, a cell surface protein known for inhibiting phagocytosis, plays a critical role in the tumor microenvironment (TME) and is a potential biomarker for cancer. However, directly applying αCD47, a hydrophilic macromolecular antibody that targets CD47, in vivo for cancer detection can have adverse effects on normal cells, cause systemic toxicities, and lead to resistance against anti-cancer therapies. In this study, we developed a novel complex incorporating aluminum-based metal-organic frameworks (Al-MOF) loaded with indocyanine green (ICG), αCD47, and resiquimod (R848), a hydrophobic small molecule Toll-like receptor 7/8 (TLR7/8) agonist. Upon activation with an infrared 808 nm laser, the nanocomposites exhibited photothermal effects that triggered the release of the loaded reagents, induced ROS production, and induced changes in the TME. This led to the polarization of immune-suppressive M2 macrophages towards an immune-stimulatory M1 phenotype, promoted dendritic cell (DC) maturation, and enabled mature DCs to facilitate antigen presentation, T-cell activation, and critical roles in tumor immunity. Furthermore, in vivo imaging successfully detected the specific binding of αCD47 with CD47 on tumor cells. Overall, the complex composed of αCD47 antibody and toll-like receptor agonist showed promising efficacy in both tumor diagnosis and therapy, providing a potential strategy for detecting early lung cancer and modulating the tumor microenvironment for improved treatment outcomes.

Dengue NS1 antibodies are associated with clearance of viral NS1.

Dengue vascular permeability syndrome is the primary cause of death in severe dengue infections. The protective versus potentially pathogenic role of dengue NS1 antibodies are not well understood. The main goal of this analysis was to characterize the relationship between free NS1 concentration and NS1 antibody titers in primary and secondary dengue infection in order to better understand the presence and duration of NS1 antibody complexes in clinical dengue infections. Hospitalized participants with acute dengue infection were recruited from Northern Colombia between 2018 to 2020. Symptom assessment including dengue signs and symptoms, chart review and blood collection was performed. Primary versus secondary Dengue was assessed serologically. NS1 titers and anti-NS1 antibodies were measured daily. Patients with secondary infection have higher antibody titers than those in primary infection, and we find a negative correlation between anti-NS1 antibody titer and NS1 protein. We demonstrate that in a subset of secondary infection, there are indeed NS1 antibody-antigen complexes at the admission day during the febrile phase that are not detectable by the recovery phase. Furthermore, dengue infection status is associated with higher circulating sialidases. The negative correlation between antibody and protein suggests that antibodies may play a role in clearing this viral protein.

The therapeutic potential of interleukin‐2/anti‐interleukin‐2 antibody complex in cold storage‐associated kidney transplantation

The therapeutic potential of interleukin‐2/anti‐interleukin‐2 antibody complex in cold storage‐associated kidney transplantation

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

CD2 costimulation as a method to improve mouse T cell activation by soluble antibody complexes

Abstract Immunology research using mouse T cells requires cell activation that is often achieved using anti-CD3/CD28 antibodies immobilized (imm) on beads or cultureware. This immobilization can be tedious, leading us to investigate mouse T cell activation using soluble (sol) antibody complexes. Purified mouse T cells were activated by immCD3/CD28 or solCD3/CD28 complexes for 2 days, resulting in an average of 85% and 68% CD25+ T cells by flow cytometry, respectively (n = 12). However, adding CD2 to the solCD3/CD28 complex increased the %CD25+ T cells to 99% after 2 days (n = 12), suggesting that CD2 enhances activation. Purified T cells activated by solCD3/CD28/CD2 complexes showed improved fold expansion (28-fold) after 7 - 8 days compared to activation by immCD3/CD28 (15-fold) or solCD3/CD28 (18-fold) complexes (n = 17). As well, purified CD4+ T cells activated by solCD3/CD28 complexes exhibited increased fold expansion from 1- to 20-fold after 8 days by adding CD2 to the complexes (n = 3). Lastly, we investigated how activation methods impact a 6-day polarization of naive CD4+ T cells to T helper subsets. Cells activated by solCD3/CD28 complexes resulted in 53% Th1, 29% Th2, or 67% Treg cells, whereas solCD3/CD28/CD2 complexes resulted in 77% Th1, 24% Th2, or 79% Treg cells (n = 6). In summary, these soluble ImmunoCult™ Mouse T Cell Activators effectively activate, expand, and polarize mouse T cell subsets, which is enhanced by CD2 costimulation.

Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107–2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Development of an immunochromatographic test strip to detect pneumolysin by monoclonal antibody capture method

BackgroundThe currently available methods for the detection of Streptococcus pneumoniae are labour-intensive, and involve time-consuming steps needing skilled technicians to perform the assays. Therefore, we developed a rapid lateral flow based immunochromatographic test strip for detection of S. pneumoniae using pneumolysin using monoclonal antibody capture approach. Methodology − The strip was designed by assembling a sample pad, conjugation pad, nitrocellulose membrane and adsorbent pad supported by a plastic backing card. Optimization of control spot was done by using three different concentrations of monoclonal antibody (0.1 µg, 0.25 g and 0.5 µg). Monoclonal antibody coupled with gold nanoparticles was immobilized on conjugate pad. Recombinant pneumolysin at three different concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) was used. Diluted pneumolysin was poured onto the sample pad which migrated through the conjugate pad containing gold antibody conjugate and the antigen–antibody complex migrated to the test spot to bind to the polyclonal antibody. The remaining free antibody bound to the control spot to visualize a control spot. Results: Control spot was optimized at 0.5 µg of mouse IgG. Of three different concentrations of Pneumolysin, the spot was best visualized at 50 ng/ml Pneumolysin concentration. Conclusion: Our ICT based device could detect a commercially available recombinant Pneumolysin of S. pneumoniae. Future study is directed to validate the strip for detection of Pneumolysin using biological fluids of patients (particularly children) infected with S. pneumoniae.