Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

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.

Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

Mendelian Causes of Autoimmunity: the Lupus Phenotype.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by its large heterogeneity in terms of clinical presentation and severity. The pathophysiology of SLE involves an aberrant autoimmune response against various tissues, an excess of apoptotic bodies, and an overproduction of type-I interferon. The genetic contribution to the disease is supported by studies of monozygotic twins, familial clustering, and genome-wide association studies (GWAS) that have identified numerous risk loci. In the early 70s, complement deficiencies led to the description of familial forms of SLE caused by a single gene defect. High-throughput sequencing has recently identified an increasing number of monogenic defects associated with lupus, shaping the concept of monogenic lupus and enhancing our insights into immune tolerance mechanisms. Monogenic lupus (moSLE) should be suspected in patients with either early-onset lupus or syndromic lupus, in male, or in familial cases of lupus. This review discusses the genetic basis of monogenic SLE and proposes its classification based on disrupted pathways. These pathways include defects in the clearance of apoptotic cells or immune complexes, interferonopathies, JAK-STATopathies, TLRopathies, and T and B cell dysregulations.

Leaky gut, circulating immune complexes, arthralgia, and arthritis in IBD: coincidence or inevitability?

Most host-microbiota interactions occur within the intestinal barrier, which is essential for separating the intestinal epithelium from toxins, microorganisms, and antigens in the gut lumen. Gut inflammation allows pathogenic bacteria to enter the blood stream, forming immune complexes which may deposit on organs. Despite increased circulating immune complexes (CICs) in patients with inflammatory bowel disease (IBD) and discussions among IBD experts regarding their potential pathogenic role in extra-intestinal manifestations, this phenomenon is overlooked because definitive evidence demonstrating CIC-induced extra-intestinal manifestations in IBD animal models is lacking. However, clinical observations of elevated CICs in newly diagnosed, untreated patients with IBD have reignited research into their potential pathogenic implications. Musculoskeletal symptoms are the most prevalent extra-intestinal IBD manifestations. CICs are pivotal in various arthritis forms, including reactive, rheumatoid, and Lyme arthritis and systemic lupus erythematosus. Research indicates that intestinal barrier restoration during the pre-phase of arthritis could inhibit arthritis development. In the absence of animal models supporting extra-intestinal IBD manifestations, this paper aims to comprehensively explore the relationship between CICs and arthritis onset via a multifaceted analysis to offer a fresh perspective for further investigation and provide novel insights into the interplay between CICs and arthritis development in IBD.

Biomimetic immobilization of α-glucosidase inspired by antibody-antigen specific recognition for catalytic preparation of 4-methylumbelliferone.

Immobilization technology plays an important role in enhancing enzyme stability and environmental adaptability. Despite its rapid development, this technology still encounters many challenges such as enzyme leakage, difficulties in large-scale implementation, and limited reusability. Drawing inspiration from natural paired molecules, this study aimed to establish a method for immobilized α-glucosidase using artificial antibody-antigen interaction. The proposed method consists of three main parts: synthesis of artificial antibodies, synthesis of artificial antigens, and assembly of the artificial antibody-antigen complex. The critical step in this method involves selecting a pair of structurally similar compounds: catechol as a template for preparing artificial antibodies and protocatechualdehyde for modifying the enzyme to create the artificial antigens. By utilizing the same functional groups in these compounds, specific recognition of the antigen by the artificial antibody can be achieved, thereby immobilizing the enzymes. The results demonstrated that the immobilization amount, specific activity, and enzyme activity of the immobilized α-glucosidase were 25.09 ± 0.10 mg/g, 5.71 ± 0.17 U/mgprotein and 143.25 ± 1.71 U/gcarrier, respectively. The immobilized α-glucosidase not only exhibited excellent reusability but also demonstrated remarkable performance in catalyzing the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside.

A link between circulating immune complexes and acute kidney injury in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum. Clinically, VL evolves with systemic impairment, immunosuppression and hyperactivation with hypergammaglobulinemia. Although renal involvement has been recognized, a dearth of understanding about the underlying mechanisms driving acute kidney injury (AKI) in VL remains. We aimed to evaluate the involvement of immunoglobulins (Igs) and immune complexes (CIC) in the occurrence of AKI in VL patients. Fourteen VL patients were evaluated between early treatment and 12 months post-treatment (mpt). Anti-Leishmania Igs, CIC, cystatin C, C3a and C5a were assessed and correlated with AKI markers. Interestingly, high levels of CIC were observed in VL patients up to 6 mpt. Concomitantly, twelve patients met the criteria for AKI, while high levels of cystatin C were observed up to 6 mpt. Plasmatic cystatin C was positively correlated with CIC and Igs. Moreover, C5a was correlated with cystatin C, CIC and Igs. We did not identify any correlation between amphotericin B use and kidney function markers in VL patients, although this association needs to be further explored in subsequent studies. Our data reinforce the presence of an important renal function impairment during VL, suggesting the involvement of Igs, CIC, and C5a in this clinical condition.

Interleukin-2/anti-interleukin-2 immune complex attenuates cold ischemia-reperfusion injury after kidney transplantation by increasing renal regulatory T cells.

BACKGROUND: Cold ischemia-reperfusion injury (IRI) is an unavoidable complication of kidney transplantation. We investigated the role of regulatory T cells (Treg) in cold IRI and whether the interleukin (IL)-2/anti-IL-2 antibody complex (IL-2C) can ameliorate cold IRI. METHODS: We developed a cold IRI mouse model using kidney transplantation and analyzed the IL-2C impact on cold IRI in acute, subacute and chronic phases. RESULTS: Treg transfer attenuated cold IRI, while Treg depletion aggravated cold IRI. Next, IL-2C administration prior to IRI mitigated acute renal function decline, renal tissue damage and apoptosis and inhibited infiltration of effector cells into kidneys and pro-inflammatory cytokine expression on day 1 after IRI. On day 7 after IRI, IL-2C promoted renal regeneration and reduced subacute renal damage. Furthermore, on day 28 following IRI, IL-2C inhibited chronic fibrosis. IL-2C decreased reactive oxygen species-mediated injury and improved antioxidant function. When IL-2C was administered following IRI, it also increased renal regeneration with Treg infiltration and suppressed renal fibrosis. In contrast, Treg depletion in the presence of IL-2C eliminated the positive effects of IL-2C on IRI. CONCLUSION: Tregs protect kidneys from cold IRI and IL-2C inhibited cold IRI by increasing the renal Tregs, suggesting a potential of IL-2C in treating cold IRI. KEY POINTS: Interleukin (IL)-2/anti-IL-2 antibody complex attenuated acute renal injury, facilitated subacute renal regeneration and suppressed chronic renal fibrosis after cold ischemia-reperfusion injury (IRI) by increasing the renal Tregs. IL-2/anti-IL-2 antibody complex decreased reactive oxygen species-mediated injury and improved antioxidant function. This study suggests the therapeutic potential of the IL-2/anti-IL-2 antibody complex in kidney transplantation-associated cold IR.

APDS patients with immune-complex vasculitis and resolution with leniolisib.

Activated phosphoinositide 3-kinase delta syndrome (APDS) is an inborn error of immunity with heterogeneous clinical manifestations of infections, immune dysregulation, autoimmunity; lymphoproliferation; and malignancy. Immune complex-mediated vasculitides have not yet been described in APDS patients. Here we offer a case series of three patients with APDS who have refractory IgA vasculitis (also called Henoch-Schönlein purpura), a form of immune complex-mediated vasculitis that activates complement and attracts neutrophils, macrophages and eosinophils to cause local tissue injury. Leniolisib is an inhibitor of PI3K p110δ and an FDA-approved treatment for APDS. IgA vasculitis resolved upon treatment with leniolisib. Patients with immune dysregulation including IgA vasculitis should be screened for APDS.

Biodistribution of Drug/ADA Complexes: The Impact of Immune Complex Formation on Antibody Distribution.

The clinical use of therapeutic monoclonal antibodies (mAbs) for the treatment of cancer, inflammation, and other indications has been successfully established. A critical aspect of drug-antibody pharmacokinetics is immunogenicity, which triggers an immune response via an anti-drug antibody (ADA) and forms drug/ADA immune complexes (ICs). As a consequence, there may be a reduced efficacy upon neutralization by ADA or an accelerated drug clearance. It is therefore important to understand immunogenicity in biological therapies. A drug-like immunoglobulin G (IgG) was radiolabeled with tritium, and ICs were formed using polyclonal ADA, directed against the complementary-determining region of the drug-IgG, to investigate in vivo biodistribution in rodents. It was demonstrated that 65% of the radioactive IC dose was excreted within the first 24 h, compared with only 6% in the control group who received non-complexed 3H-drug. Autoradiographic imaging at the early time point indicated a deposition of immune complexes in the liver, lung, and spleen indicated by an increased radioactivity signal. A biodistribution study confirmed the results and revealed further insights regarding excretion and plasma profiles. It is assumed that the immune complexes are readily taken up by the reticuloendothelial system. The ICs are degraded proteolytically, and the released radioactively labeled amino acids are redistributed throughout the body. These are mainly renally excreted as indicated by urine measurements or incorporated into protein synthesis. These biodistribution studies using tritium-labeled immune complexes described in this article underline the importance of understanding the immunogenicity induced by therapeutic proteins and the resulting influence on biological behavior.

[Examination of Urinary Sediment in a Patient with Lupus-Like HIV-Associated Immune Complex Kidney Disease (HIVICK) - Case Report and Review of the Literature].

Renal involvement is very common in patients with HIV infection. The phenotype varies from the most frequently "collapsing" variant of focal and segmental glomerulosclerosis (FSGS) to "lupus-like HIV-immune complex kidney disease" (HIVICK). The latter is characterized by a histological picture that recalls lupus nephropathy. Through a clinical case, we underline the importance of urinary sediment analysis in patients with suspected glomerulopathy. Findings such as the characteristic cells that show the typical appearance of Herpes virus (HSV) infection or LE cells have significantly supported the diagnosis of HIVICK. In light of the present observations, we suggest systematically carrying out a cytological examination of the urinary sediment to confirm diagnostic hypotheses of rare pathologies.

AbFlex: designing antibody complementarity determining regions with flexible CDR definition.

MOTIVATION: Antibodies are proteins that the immune system produces in response to foreign pathogens. Designing antibodies that specifically bind to antigens is a key step in developing antibody therapeutics. The complementarity determining regions (CDRs) of the antibody are mainly responsible for binding to the target antigen, and therefore must be designed to recognize the antigen. RESULTS: We develop an antibody design model, AbFlex, that exhibits state-of-the-art performance in terms of structure prediction accuracy and amino acid recovery rate. Furthermore, >38% of newly designed antibody models are estimated to have better binding energies for their antigens than wild types. The effectiveness of the model is attributed to two different strategies that are developed to overcome the difficulty associated with the scarcity of antibody-antigen complex structure data. One strategy is to use an equivariant graph neural network model that is more data-efficient. More importantly, a new data augmentation strategy based on the flexible definition of CDRs significantly increases the performance of the CDR prediction model. AVAILABILITY AND IMPLEMENTATION: The source code and implementation are available at https://github.com/wsjeon92/AbFlex.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

Background: The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Methods: Six-week-old wildtype (WT+ONA) and ßB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. Results: We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. Conclusion: These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Glucocorticoid-free remission in patients with SLE in the era of biologics: Immune complex disease is likely to benefit from current medications.

OBJECTIVES: In addition to various immunosuppressive agents, belimumab and anifrolumab became available in Japan. We aimed to investigate glucocorticoid-free clinical remission in a single-centre retrospective cohort in October 2023. METHODS: Our cohort included patients with SLE who needed to start or increase glucocorticoids for disease activity and were followed up for more than 1 year. We investigated the rate of achievement of clinical remission off corticosteroids (CR off C), defined as no clinical score on the SLEDAI-2K without glucocorticoids, baseline predictors of CR off C, medications used when CR off C was achieved, and flare rates following CR off C. RESULTS: Out of the 60 patients followed for an average of 5.4 (±2.6) years, 17 (28.3%) achieved CR off C in 3.6 (±1.2) years after enrolment. Use of belimumab and anifrolumab accounted for eight (47.1%) of the achievers. Among the baseline data, male sex, recent enrolment, high glucocorticoid dose, and detection of immune complex (IC) significantly predicted CR off C, while lupus nephritis (LN) and a low C3 level tended to predict it. In the multivariate analysis, IC detection was the only predictor of CR off C. Clinical flares were observed in 5.9% of the achievers during a median 1.2 years after achievement of CR off C. CONCLUSION: In the era of biologics, CR off C was achieved in 28.3% of the patient cohort requiring the start or increase of glucocorticoids for disease activity, with a relatively low rate of flares, suggesting that glucocorticoid-free clinical remission is an achievable target in SLE. IC disease, represented by male sex or nephritis, is likely to benefit from currently available medications.

Molecular architecture and platelet-activating properties of small immune complexes assembled on heparin and platelet factor 4.

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents. Native mass spectrometry demonstrates that up to three PF4 tetramers can be assembled on a heparin chain, consistent with the molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

Exploring the Link between Hydrodynamic Size and Immunoglobulins of Circulating Immune Complexes in Rheumatoid Arthritis Patients.

The function of immune complexes in rheumatoid arthritis (RA) is related to their composition and size. Using dynamic light scattering (DLS), we investigated the link between the RA circulating immune complex (CIC) particles' size and the CIC immunoglobulin level. In this study, 30 RA patients and 30 healthy individuals were included. IgA, IgG, and IgM were found in all analyzed CICs, but more IgA and IgG were found in RA than in control CICs. In both control and RA CICs, DLS detected 50 particles that differed in size and clustered around two size groups: with a 7.5-164 nm radius and with a 342-1718 nm radius. An increased level of IgA in RA CICs, compared to control ones, was associated with more than 50% of CIC particles. In RA, compared to the control, a higher number of CICs with 28.2 nm, 531 nm, 712 nm, and 1718 nm particles and a lower number of CICs with 78.8 nm particles were detected. This particle distribution pattern did not reflect the changes in the CIC immunoglobulin level. Thus, RA elevated CIC IgA was linked with all these particles (except the 1718 nm particle), the IgM increase was linked with 43.8 nm and 712 nm particles, and the IgG increase was linked with the 712 nm particle only. This study provides the very first data on the association between CIC particles' size, CIC immunoglobulin level, and RA. It opens the possibility that the size of CICs determined by DLS can be used as a criterion in RA diagnosis or monitoring after a large-scale study confirmation.

ThermoPCD: a database of molecular dynamics trajectories of antibody-antigen complexes at physiologic and fever-range temperatures.

Progression of various cancers and autoimmune diseases is associated with changes in systemic or local tissue temperatures, which may impact current therapies. The role of fever and acute inflammation-range temperatures on the stability and activity of antibodies relevant for cancers and autoimmunity is unknown. To produce molecular dynamics (MD) trajectories of immune complexes at relevant temperatures, we used the Research Collaboratory for Structural Bioinformatics (RCSB) database to identify 50 antibody:antigen complexes of interest, in addition to single antibodies and antigens, and deployed Groningen Machine for Chemical Simulations (GROMACS) to prepare and run the structures at different temperatures for 100-500 ns, in single or multiple random seeds. MD trajectories are freely available. Processed data include Protein Data Bank outputs for all files obtained every 50 ns, and free binding energy calculations for some of the immune complexes. Protocols for using the data are also available. Individual datasets contain unique DOIs. We created a web interface, ThermoPCD, as a platform to explore the data. The outputs of ThermoPCD allow the users to relate thermally-dependent changes in epitopes:paratopes interfaces to their free binding energies, or against own experimentally derived binding affinities. ThermoPCD is a free to use database of immune complexes' trajectories at different temperatures that does not require registration and allows for all the data to be available for download. Database URL: https://sites.google.com/view/thermopcd/home.

Sparsentan ameliorates glomerular hypercellularity and inflammatory-gene networks induced by IgA1-IgG immune complexes in a mouse model of IgA nephropathy.

IgA nephropathy (IgAN) is characterized by glomerular deposition of immune complexes (ICs) consisting of IgA1 with O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These ICs induce kidney injury, and in the absence of disease-specific therapy, up to 40% of patients with IgAN progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. Here, we used a model wherein engineered ICs (EICs) formed from human Gd-IgA1 and recombinant human IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. In this model, we assessed the protective effects of sparsentan, a single-molecule dual endothelin angiotensin receptor antagonist (DEARA) versus vehicle on EIC-induced glomerular proliferation and dysregulation of gene expression in the kidney. Oral administration of sparsentan (60 or 120 mg/kg daily) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity. Furthermore, analysis of changes in the whole kidney transcriptome revealed that key inflammatory and proliferative biological genes and pathways that are upregulated in this EIC model of IgAN were markedly reduced by sparsentan, including complement genes, integrin components, members of the mitogen-activated protein kinase family, and Fc receptor elements. Partial overlap between mouse and human differentially expressed genes in IgAN further supported the translational aspect of the immune and inflammatory components from our transcriptional findings. In conclusion, our data indicate that in the mouse model of IgAN, sparsentan targets immune and inflammatory processes leading to protection from mesangial hypercellularity.NEW & NOTEWORTHY The mechanisms by which deposited IgA1 immune complexes cause kidney injury during early phases of IgA nephropathy are poorly understood. We used an animal model we recently developed that involves IgA1-IgG immune complex injections and determined pathways related to the induced mesangioproliferative changes. Treatment with sparsentan, a dual inhibitor of endothelin type A and angiotensin II type 1 receptors, ameliorated the induced mesangioproliferative changes and the associated alterations in the expression of inflammatory genes and networks.

Do antibody CDR loops change conformation upon binding?

Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.

In situ detection of silk fibroin using a dual recognition strategy with a flexible pressure immunosensor.

Silk is a symbol of ancient Chinese civilization that has made an indelible contribution to the development of world civilization. However, because ancient artifacts are often contaminated or degraded, it is difficult to detect the presence of silk therein, and the true origin of silk thus remains a mystery. Therefore, this work presents a flexible pressure immunosensor that was designed based on 3D polypyrrole (PPy) foams for the trace detection of silk fibroin at archaeological sites. Initially, silk fibroin (SF) was conjugated with antibody-functionalized copper oxide nanoparticles (CuO NPs) and carboxylated magnetic beads (MBs) to form a sandwich immune complex. Then, the sandwich immune complex was added to hydrogen peroxide (H2O2) by magnetic separation to catalyse the generation of oxygen (O2), which converted the antigen-antibody specific recognition signal to gas pressure. As the pressure within the device increases, the 3D PPy foam, as the sensing layer resistance was 150 Ω, undergoes extrusion and deformation. This deformation leads to alterations in the foam resistance. The flexible pressure immunosensor can sensitively monitor the change in electrical resistance in the system and quantitatively detect silk fibroin. With optimization, the flexible pressure immunosensor demonstrates a dynamic range of operation spanning from 10 ng mL-1 to 100 µg mL-1, exhibiting a remarkable detection limit of 10.58 ng mL-1 specifically for silk fibroin. Notably, this immunosensor surpasses enzyme-linked immunosorbent assay (ELISA) in terms of superior reproducibility, specificity, and accuracy. Therefore, this application provides a new method and technical support for silk detection.