Ab Complex Research Articles

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.

IL-4/IL-4 Ab complex enhances the accumulation of both antigen-specific and bystander CD8 T cells in mouse lungs infected with influenza A virus

BackgroundUnlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection.ResultsIn vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3.ConclusionsThese data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs.

Evaluation of the Ability of AlphaFold to Predict the Three-Dimensional Structures of Antibodies and Epitopes.

Being able to accurately predict the three-dimensional structure of an Ab can facilitate Ab characterization and epitope prediction, with important diagnostic and clinical implications. In this study, we evaluated the ability of AlphaFold to predict the structures of 222 recently published, high-resolution Fab H and L chain structures of Abs from different species directed against different Ags. We show that although the overall Ab prediction quality is in line with the results of CASP14, regions such as the complementarity-determining regions (CDRs) of the H chain, which are prone to higher variation, are predicted less accurately. Moreover, we discovered that AlphaFold mispredicts the bending angles between the variable and constant domains. To evaluate the ability of AlphaFold to model Ab-Ag interactions based only on sequence, we used AlphaFold-Multimer in combination with ZDOCK to predict the structures of 26 known Ab-Ag complexes. ZDOCK, which was applied on bound components of both the Ab and the Ag, succeeded in assembling 11 complexes, whereas AlphaFold succeeded in predicting only 2 of 26 models, with significant deviations in the docking contacts predicted in the rest of the molecules. Within the 11 complexes that were successfully predicted by ZDOCK, 9 involved short-peptide Ags (18-mer or less), whereas only 2 were complexes of Ab with a full-length protein. Docking of modeled unbound Ab and Ag was unsuccessful. In summary, our study provides important information about the abilities and limitations of using AlphaFold to predict Ab-Ag interactions and suggests areas for possible improvement.

Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8+ T cells against tumor

BackgroundA key to success of cancer immunotherapy is the amplification and sustenance of various effector cells. The hallmark of prominent antitumor T cells is their long-term effector function. Although interleukin...

Single-molecule investigations of single-chain cellulose biosynthesis

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

A method for Boolean analysis of protein interactions at a molecular level

Determining the levels of protein–protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean – a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets.

Antibody recruiting molecules (ARMs) represent an important class of "proximity-inducing" chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten-specific serum antibody (Ab) (e.g., anti-dinitrophenyl (DNP)) and a cancer cell surface protein, enforcing their proximity. ARM anticancer efficacy depends on the formation of ARM:Ab complexes on the cancer cell surface, which activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes, namely, intrinsically low binding affinity and/or low concentrations of anti-hapten antibodies in human serum. To address this potential limitation, we previously developed a covalent ARM (cARM) chemical tool that eliminates the ARM:antibody equilibrium through a covalent linkage. In the current study, we set out to determine to what extent maximizing the stability of ARM:antibody complexes via cARMs enhances target immune recognition. We observe cARMs significantly increase target immune recognition relative to ARMs across a range of therapeutically relevant antibody concentrations. These results demonstrate that ARM therapeutic function can be dramatically enhanced by increasing the kinetic stability of ARM:antibody complexes localized on cancer cells. Our findings suggest that a) high titres/concentrations of target antibody in human serum are not neccessary and b) saturative antibody recruitment to cancer cells not sufficient, to achieve maximal ARM therapeutic function.

Tumor suppressor lnc-CTSLP4 inhibits EMT and metastasis of gastric cancer by attenuating HNRNPAB-dependent Snail transcription

Tumor metastasis is a crucial impediment to the treatment of gastric cancer (GC), and the epithelial-to-mesenchymal transition (EMT) program plays a critical role for the initiation of GC metastasis. Thus, the aim of this study is to investigate the regulation of lnc-CTSLP4 in the EMT process during GC progression. We found that lnc-CTSLP4 was significantly downregulated in GC tumor tissues compared with adjacent non-tumor tissues, and its levels in GC tumor tissues were closely correlated with tumor local invasion, TNM stage, lymph node metastasis, and prognosis of GC patients. Loss- and gain-of-function assays indicated that lnc-CTSLP4 inhibited GC cell migration, invasion, and EMT in vitro, as well as peritoneal dissemination in vivo. Mechanistic analysis demonstrated that lnc-CTSLP4 could bind with Hsp90α/heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) complex and recruit E3-ubiquitin ligase ZFP91 to induce the degradation of HNRNPAB, thus suppressing the transcriptional activation of Snail and ultimately reversing EMT of GC cells. Taken together, our results suggest that lnc-CTSLP4 is significantly downregulated in GC tumor tissues and inhibits metastatic potential of GC cells by attenuating HNRNPAB-dependent Snail transcription via interacting with Hsp90α and recruiting E3 ubiquitin ligase ZFP91, which shows that lnc-CTSLP4 could serve as a prognostic biomarker and therapeutic target for metastatic GC.

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets

Antibody recruiting molecules (ARMs) represent an important class of chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten specific serum antibody (Ab) and a cancer cell surface protein, thereby forming ARM:Ab complexes on the cancer cell surface. These complexes activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes; namely intrinsically low binding affinity of anti-hapten antibodies in human serum. To address this potential limitation, we applied covalent ARM (cARM) chemical tools to eliminate the ARM:Ab equilibrium through a covalent linkage. In the current study we determine to what extent stabilizing ARM:Ab complexes via cARMs enhances target immune recognition. We observe cARMs significantly enhance immune function relative to ARMs and demonstrate ARM therapeutic function can be dramatically enhanced by increasing the stability of ARM:antibody complexes on cancer cells.

OxLDL/β2GPI/anti‑β2GPI Ab complex induces inflammatory activation via the TLR4/NF‑κB pathway in HUVECs.

Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti-β2-glycoprotein I (β2GPI) antibody (Ab). Our previous studies revealed that the anti-β2GPI Ab formed a stable oxidized low-density lipoprotein (oxLDL)/β2GPI/anti-β2GPI Ab complex, which accelerated AS development by promoting the accumulation of lipids in macrophages and vascular smooth muscle cell. However, the effects of the complex on endothelial cells, which drive the initiation and development of AS, remain unknown. Thus, the present study aimed to determine the proinflammatory roles of the oxLDL/β2GPI/anti-β2GPI Ab complex in human umbilical vein endothelial cells (HUVECs) in an attempt to determine the underlying mechanism. Reverse transcription-quantitative PCR, enzymy-linked immunosorbent assay, western blotting and immunofluorescence staining were performed to detect the expressions of inflammation related factors and adhesion molecules. Monocyte-binding assay was used to investigate the effects of oxLDL/β2GPI/anti-β2GPI Ab complex on monocyte adhesion to endothelial cells. The results demonstrated that the oxLDL/β2GPI/anti-β2GPI Ab complex upregulated the expression of Toll-like receptor (TLR)4 and the levels of NF-κB phosphorylation in HUVECs, and subsequently enhanced the expression levels of inflammatory cytokines, including TNF-α, IL-1β and IL-6, as well as those of adhesion molecules, such as intercellular adhesion molecule 1 and vascular adhesion molecule 1. In addition, the complex facilitated the recruitment of monocytes by promoting the secretion of monocyte chemotactic protein 1 in HUVECs. Notably, the described effects of the oxLDL/β2GPI/anti-β2GPI Ab complex in HUVECs were abolished by either TLR4 or NF-κB blockade. In conclusion, these findings suggested that the oxLDL/β2GPI/anti-β2GPI Ab complex may induce a hyper-inflammatory state in endothelial cells by promoting the secretion of proinflammatory cytokines and monocyte recruitment, which was discovered to be largely dependent on the TLR4/NK-κB signaling pathway.

Impaired host response and the presence of Acinetobacter baumannii in the serum microbiome of type-II diabetic patients.

SummaryType II diabetes (T2D) affects over 10% of the US population and is a growing disease worldwide that manifests with numerous comorbidities and defects in inflammation. This dysbiotic host response allows for infection of the host by numerous microorganisms. In the course of T2D disease, individuals can develop chronic infections including foot ulcers and periodontitis, which lead to further complications and opportunistic infections in multiple body sites. In this study, we investigated the serum of healthy subjects and patients with T2D with (T2DP) or without periodontitis for both microbiome signatures in addition to cytokine profiles. Surprisingly, we detected the presence of Acinetobacter baumanii in the serum of 23% individuals with T2D/T2DP tested. In T2DP, IL-1β, TNF-α, MCP-1, IL-6, IL-8, and IFN-γ were significantly elevated in ABC-positive subjects. As an emerging pathogen, A. baumanii infection represents a risk for impaired inflammation and the development of comorbidities in subjects with T2D.

Label-free electrochemical immunosensor for sensitive HER2 biomarker detection using the core-shell magnetic metal-organic frameworks

Abstract Early diagnosis of tumors in the early stages of cancer allows for successful treatment and complete recovery. Herein, a simple and novel electrochemical immunosensor for Human Epidermal growth factor Receptor 2 (HER2) detection was constructed employing electrocatalytic activity of the magnetic framework Fe3O4@ TMU-21. The surface of the Fe3O4 nanoparticles was functionalized with methacrylic acid (MAA) to facilitate the immobilization of TMU-21. The fabrication of the immunosensor platform is based on Fe3O4@ TMU-21, and multi-walled carbon nanotubes (MWCNT). Their electrocatalytic activity towards the reduction of hydrogen peroxide is applied to signal immunosensor. In the presence of HER2 biomarker (Ag), the amperometric current of the decline of H2O2 decreased, which could be concluded that the establishment of Ag –Ab complexes on the electrode, and consequence blocking the electron transfer of reduction of H2O2 at the electrode surface. The analytical performance results of the immunosensor indicate that a linear relationship between the logarithm of the HER2 concentrations and amperometric responses is within the range of 1.0 pg mL−1-100 ng mL−1 and the detection limit was 0.3 pg mL−1. Remarkably, the detection result of HER2 in real human serum samples by the proposed immunosensor is well in correlation with HER2 spiked to human blood serum samples, therefore applies to the recognition of HER2 biomarker in clinical monitoring of tumor marker.

Spectroscopic and insilico Studies of Ab (1-16) on Copper induced conformational changes in Poly (dG-dC). Poly ( dG-dC) [Poly(GC)] DNA sequence: Implications in Alzheimer’s disease

DNA molecules are dynamic and play a crucial role in life processes. DNA is polymorphic and flexible having the ability to undergo structural changes depending on the environment. The structural property and stability of DNA are very important for cellular functions, and are involved in normal functioning of processes like replication, repair of DNA and protein synthesis processes. Metals like Fe and Cu are known to play a crucial role in brain functioning and deviations in concentration from the normal are often recognized as symptoms of malfunctions or disease. The effect of copper(Cu) and amyloid peptide (Ab ) on DNA in the brain has been considered very important due to their role in oxidative DNA damage, structural changes of the Amyloid peptide and are the etiological factors for neurodegenerative diseases. In the current investigation, changes in conformation of Poly (GC) sequence DNA induced by copper and Ab 1-16 is studied using Circular Dichroism (CD). CD studies have shown the shift in conformation of DNA, from B form to an altered B-, and C-DNA conformation. The CD studies were substantiated by Molecular docking confirms that the Ab -copper complexes with DNA via hydrogen bond formed between Guanine-Cytosine (GC) pairs. This work gives clues as to how copper Ab complex modulates DNA conformations which attributes to the stability. Keywords: Alzheimer’s disease, DNA, Copper, Circular Dichroism, Molecular Docking.

A molecular dynamic simulation approach: development of dengue virus vaccine by affinity improvement techniques

This study is about proposing a vaccine for all four strains of dengue virus (DENV) that could be an important approach for reaching the WHO goal of reducing dengue morbidity and mortality. The significance of the DENV envelope proteins III lies in the fact that it elicits an immune response and hence can be a potential vaccine design candidate. This domain appears to play a key role in the host cell receptor binding for viral entry and in inducing long lasting protective immunity against the infection. We used long molecular dynamic simulation and mutagenesis scanning methods to provide the dynamic environment and propose the potential mutation that may result in enhancing the binding specificity and affinity of the antigen–antibody (Ag–Ab) complex. The binding free energetics were also estimated using free energy perturbation method. One charged mutation that is theorinine 93L to arginine interacting with epitopic glutamic acid 368 strongly contributing in increasing the binding affinity as well as specificity, predicted as −9.6 kcal/mol gain in 2H12-Fab with dengue envelope domain III binding free energy relative to the wild-type. In conclusion, the one charged residue that showed theoretically enhances the binding affinity of Ag–Ab complex by making couple of interactions i.e. by substituting theorinine to arginine in the antibody chains and can be considered as potential dengue vaccine candidate. Communicated by Ramaswamy H. Sarma

Morphological transformations of AB diblock copolymer particles in soft confinement

The morphologies of diblock copolymers self-assembled under selected solvent have been investigated systematically using a simulated annealing method. Various categories of internal morphologies are observed. The morphology transform can be modulated by three important factors: softness of confinement, volume fraction of A/B monomers ([Formula: see text] and [Formula: see text]) and selectivity of solvent. With increase in [Formula: see text], the selectivity of solvent can determine the type and number of cores. The softness of confinement can greatly influence the shape of copolymer which prefers to be sphere or nonsphere. Stacked lamella morphology is an efficient method to reduce the contact number between A-B blocks in softer confinement. Core–shell morphologies usually have large curly degree with small end-to-end distance of AB complex chain. The nucleation condition is that the ratio of end-to-end distance of AB complex chain to the length of whole chain is lower than 0.28. The simulation results can provide theoretical guidance for experiment in synthesis of nanoparticles.

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin-Ab complexes.

Ricin toxin is a plant‐derived, ribosome‐inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)—with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a “lactose‐sensitive” pathway mediated by ricin's galactose/N‐acetylgalactosamine‐specific lectin subunit (RTB), and a “mannose‐sensitive” pathway mediated by the mannose receptor (MR; CD206) or other C‐type lectins capable of recognizing the mannose‐side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin‐specific mouse MAb and camelid single‐domain (VHH) antibodies to protect KCs and LSECs from ricin‐induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VHHs afforded little (<40%) or even no protection to LSECs against ricin‐induced death. Complete protection of LSECs was only achieved with MAb or VHH cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose‐sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab‐based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.

The Inhibitory Effect of Celangulin V on the ATP Hydrolytic Activity of the Complex of V-ATPase Subunits A and B in the Midgut of Mythimna separata.

Celangulin V (CV) is a compound isolated from Celastrus angulatus Max that has a toxic activity against agricultural insect pests. CV can bind to subunits a, H, and B of the vacuolar ATPase (V-ATPase) in the midgut epithelial cells of insects. However, the mechanism of action of CV is still unclear. In this study, the soluble complex of the V-ATPase A subunit mutant TSCA which avoids the feedback inhibition by the hydrolysate ADP and V-ATPase B subunit were obtained and then purified using affinity chromatography. The H+K+-ATPase activity of the complex and the inhibitory activity of CV on ATP hydrolysis were determined. The results suggest that CV inhibits the ATP hydrolysis, resulting in an insecticidal effect. Additionally, the homology modeling of the AB complex and molecular docking results indicate that CV can competitively bind to the AB complex at the ATP binding site, which inhibits ATP hydrolysis. These findings suggest that the AB subunits complex is one of the potential targets for CV and is important for understanding the mechanism of interaction between CV and V-ATPase.

Rapid quantitative protein detection by light transmission spectroscopy.

Rapid, sensitive, and quantitative protein detection is critical for many applications in medicine, environmental monitoring, and the food industry. Advancements in detection of proteins include the use of antigen-antibody binding; however, many current methods are time-consuming and have limiting factors such as low sensitivity and the inability to provide absolute values. We present a new high-throughput method for protein detection using light transmission spectroscopy (LTS), which can quantify and size nanoparticles in fluid suspension. LTS can quantify proteins directly and target specific proteins through antigen-antibody binding. This work shows that LTS can distinguish between and quantify bovine serum albumin, its antibody, and the BSA + Ab complex and determine BSA protein concentrations down to 5 μg/mL. We use both Mie and discrete dipole approximation models to provide geometric insight into the binding process.

Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.