Selection Of Antibodies Research Articles

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

Sotrovimab: A Review of Its Efficacy against SARS-CoV-2 Variants.

Among the anti-Spike monoclonal antibodies (mAbs), the S-309 derivative sotrovimab was the most successful in having the longest temporal window of clinical use, showing a high degree of resiliency to SARS-CoV-2 evolution interrupted only by the appearance of the BA.2.86* variant of interest (VOI). This success undoubtedly reflects rational selection to target a highly conserved epitope in coronavirus Spike proteins. We review here the efficacy of sotrovimab against different SARS-CoV-2 variants in outpatients and inpatients, discussing both randomized controlled trials and real-world evidence. Although it could not be anticipated at the time of its development and introduction, sotrovimab's use in immunocompromised individuals who harbor large populations of variant viruses created the conditions for its eventual demise, as antibody selection and viral evolution led to its eventual withdrawal due to inefficacy against later variant lineages. Despite this, based on observational and real-world data, some authorities have continued to promote the use of sotrovimab, but the lack of binding to newer variants strongly argues for the futility of continued use. The story of sotrovimab highlights the power of modern biomedical science to generate novel therapeutics while also providing a cautionary tale for the need to devise strategies to minimize the emergence of resistance to antibody-based therapeutics.

Antibody selection strategies and their impact in predicting clinical malaria based on multi-sera data

BackgroundNowadays, the chance of discovering the best antibody candidates for predicting clinical malaria has notably increased due to the availability of multi-sera data. The analysis of these data is typically divided into a feature selection phase followed by a predictive one where several models are constructed for predicting the outcome of interest. A key question in the analysis is to determine which antibodies should be included in the predictive stage and whether they should be included in the original or a transformed scale (i.e. binary/dichotomized).MethodsTo answer this question, we developed three approaches for antibody selection in the context of predicting clinical malaria: (i) a basic and simple approach based on selecting antibodies via the nonparametric Mann–Whitney-Wilcoxon test; (ii) an optimal dychotomizationdichotomization approach where each antibody was selected according to the optimal cut-off via maximization of the chi-squared (χ2) statistic for two-way tables; (iii) a hybrid parametric/non-parametric approach that integrates Box-Cox transformation followed by a t-test, together with the use of finite mixture models and the Mann–Whitney-Wilcoxon test as a last resort. We illustrated the application of these three approaches with published serological data of 36 Plasmodium falciparum antigens for predicting clinical malaria in 121 Kenyan children. The predictive analysis was based on a Super Learner where predictions from multiple classifiers including the Random Forest were pooled together.ResultsOur results led to almost similar areas under the Receiver Operating Characteristic curves of 0.72 (95% CI = [0.62, 0.82]), 0.80 (95% CI = [0.71, 0.89]), 0.79 (95% CI = [0.7, 0.88]) for the simple, dichotomization and hybrid approaches, respectively. These approaches were based on 6, 20, and 16 antibodies, respectively.ConclusionsThe three feature selection strategies provided a better predictive performance of the outcome when compared to the previous results relying on Random Forest including all the 36 antibodies (AUC = 0.68, 95% CI = [0.57;0.79]). Given the similar predictive performance, we recommended that the three strategies should be used in conjunction in the same data set and selected according to their complexity.

Characterization of antibody surface properties and selection of a diverse subset for development of a generic size-exclusion chromatography method

Aggregates are an important quality attribute for biotherapeutics because they can affect the safety and efficacy of the drug and they are therefore routinely monitored, mainly with size-exclusion chromatography (SEC). However, there is often a need to tailor a SEC method to a specific molecule. This study describes the development of a generic SEC method tested on 138 antibodies with the variable domains taken from clinical stage antibodies. We report on the discovery of a subset of 12 antibodies that represents the full range of physiochemical properties found in the 138 antibodies. This subset is shown to be an efficient and reliable test set when developing chromatographic methods for antibodies. An understanding of the nature of the analyte-stationary phase interactions was gained when using this set with its wide range of physiochemical properties. Highly hydrophobic antibodies interact strongly with some modern silica hybrid materials causing the elution time to increase significantly, while a hydrophilically modified hybrid surface showed highly reduced interactions for the hydrophobic antibodies. Highly hydrophilic antibodies, on the other hand, exhibited asymmetric peaks to a certain extent on all stationary phases, while the elution time was not affected. The developed SEC method was shown to have satisfactory performance in terms of linearity, repeatability, range, and accuracy and exhibit very narrow distributions of elution time and peak symmetry when testing the 138 antibodies indicating its generic performance.

Antibody production relies on the tRNA inosine wobble modification to meet biased codon demand.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.

High-resolution map of the Fc functions mediated by COVID-19-neutralizing antibodies.

A growing body of evidence shows that fragment crystallizable (Fc)-dependent antibody effector functions play an important role in protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To unravel the mechanisms that drive these responses, we analyzed the phagocytosis and complement deposition mediated by a panel of 482 human monoclonal antibodies (nAbs) neutralizing the original Wuhan virus, expressed as recombinant IgG1. Our study confirmed that nAbs no longer neutralizing SARS-CoV-2 Omicron variants can retain their Fc functions. Surprisingly, we found that nAbs with the most potent Fc function recognize the N-terminal domain, followed by those targeting class 3 epitopes in the receptor binding domain. Interestingly, nAbs direct against the class 1/2 epitopes in the receptor binding motif, which are the most potent in neutralizing the virus, were the weakest in Fc functions. The divergent properties of the neutralizing and Fc function-mediating antibodies were confirmed by the use of different B cell germlines and by the observation that Fc functions of polyclonal sera differ from the profile observed with nAbs, suggesting that non-neutralizing antibodies also contribute to Fc functions. These data provide a high-resolution picture of the Fc-antibody response to SARS-CoV-2 and suggest that the Fc contribution should be considered for the design of improved vaccines, the selection of therapeutic antibodies, and the evaluation of correlates of protection.

Exploring the Sensitivity of Antibody-Drug Conjugate Efficacy to the Selection of Payload, Antibody, and Cell line.

Antibody-drug conjugates (ADCs) make up a growing class of targeted therapeutics with important applications in cancer treatment. ADCs are highly modular in nature and thus can be engineered to target any cancer type, but their efficacy is strongly influenced by the specific choice of payload, antibody, and target cell. Considering the number of possible antibody-payload combinations, ADC development would benefit from an efficient method to narrow the number of ADC compositions to those with the highest and most universal potency prior to assessing pharmacokinetics and pharmacodynamics in animal models. To facilitate the identification of optimal ADC compositions, we describe the use of photoreactive antibody-binding domain-drug conjugates (known commercially as oYo-Link) to enable the site-specific labeling of off-the-shelf antibodies. This approach allows for the rapid generation of ADCs with a drug-to-antibody ratio of ∼2 with no subsequent purification required. As a demonstration of this approach, ADCs were generated with different combinations of tubulin-inhibitor drugs (DM1, DM4, VcMMAE, and VcMMAF) and anti-EGFR antibodies (cetuximab, panitumumab, anti-EGFR clone 425, and anti-EGFR clone 528) and were delivered to three EGFR-expressing cell lines (A431, A549, and MDA-MB-231). Real-time cytolysis assays indicated that the most effective antibody varied based on the choice of cell line: cetuximab was most potent against A431 cells, while 425 and 528 led to the greatest cytotoxicity against A549 and MDA-MB-231 cells. These results did not correlate with differences in measured anti-EGFR binding affinity as cetuximab had the highest affinity across all three cell lines, while 425 and 528 had the lowest affinities for all three cell lines. Panitumumab, which had the second-highest anti-EGFR affinity, exhibited the least effective cytolysis across A431, A549, and MDA-MB-231 cells. By demonstrating that ADC potency toward a given target is dependent on both the antibody and drug chosen, these findings can guide the selection of ADCs for further in vivo analysis.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Antigen density and applied force control enrichment of nanobody-expressing yeast cells in microfluidics.

In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics.

Antibodies, repertoires and microdevices in antibody discovery and characterization.

Therapeutic antibodies are paramount in treating a wide range of diseases, particularly in auto-immunity, inflammation and cancer, and novel antibody candidates recognizing a vast array of novel antigens are needed to expand the usefulness and applications of these powerful molecules. Microdevices play an essential role in this challenging endeavor at various stages since many general requirements of the overall process overlap nicely with the general advantages of microfluidics. Therefore, microfluidic devices are rapidly taking over various steps in the process of new candidate isolation, such as antibody characterization and discovery workflows. Such technologies can allow for vast improvements in time-lines and incorporate conservative antibody stability and characterization assays, but most prominently screenings and functional characterization within integrated workflows due to high throughput and standardized workflows. First, we aim to provide an overview of the challenges of developing new therapeutic candidates, their repertoires and requirements. Afterward, this review focuses on the discovery of antibodies using microfluidic systems, technological aspects of micro devices and small-scale antibody protein characterization and selection, as well as their integration and implementation into antibody discovery workflows. We close with future developments in microfluidic detection and antibody isolation principles and the field in general.

Recent clinical evidence on nutrition, novel pharmacotherapy, and vaccination in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), which enclose Crohn's disease (CD) and ulcerative colitis (UC), are chronic, relapsing inflammatory ailments. Their specific pathogenesis is not completely clarified, the worldwide incidence and prevalence of IBD has been steadily growing, and there is still not a definitive cure. The management of IBD has become more and more targeted, with specific immune mediators identified to be involved in its pathogenesis. Vedolizumab, a humanised monoclonal antibody binding specifically to the α4β7 integrin, is a gut-selective immunosuppressive biologic drug administered for both CD and UC. With the same indications as vedolizumab, ustekinumab is a fully human IgG1κ monoclonal antibody binding with specificity to the shared p40 protein subunit of human cytokines interleukin (IL)-12 and IL-23. Several selective IL-23p19 monoclonal antibodies (risankizumab, mirikizumab, and guselkumab) have also revealed admirable efficacy and safety in IBD patients. Nutrition is a very important environmental factor associated with the onset and progression of IBD, and the Western diet is considered to contribute to the development of IBD. In this narrative review, our aim is to present an overview of the main results from recent clinical studies on IBD regarding diet, new drug treatments, and also vaccination.

Quantitative determination of prothioconazole in wheat grain, soybean, and pond water based on a polyclonal antibody.

Prothioconazole and its metabolite are considered a potential threat to human health and environmental safety. Thus, the development of a sensitive and rapid detection method for prothioconazole is crucial to ensure the safety of agricultural products. In this study, a new hapten of prothioconazole was designed and synthesized, and a selective polyclonal antibody with high affinity against prothioconazole was produced, which was obtained from immunized New Zealand white rabbits. Based on the polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) were developed for detecting prothioconazole pesticides. Under optimized experimental conditions, the limit of quantification (LOQ) values for ic-CLEIA and ic-ELISA were 1.8 and 10.7 ng mL-1, respectively. The results demonstrated that the sensitivity (LOQ) achieved by ic-CLEIA was more than five times higher compared to that obtained with ic-ELISA. In addition, the recoveries obtained by adding standard prothioconazole to wheat grain, soybean, and pond water samples were in the range of 81.9 to 104.7% for ic-ELISA and 89.0 to 118.0% for ic-CLEIA.

Review on anti-alzheimer drug development: approaches, challenges and perspectives.

Alzheimer is an irreversible progressive neurodegenerative disease that causes failure of cerebral neurons and disability of the affected person to practice normal daily life activities. There is no concrete evidence to identify the exact reason behind the disease, so several relevant hypotheses emerged, highlighting many possible therapeutic targets, such as acetylcholinesterase, cholinergic receptors, N-methyl d-aspartate receptors, phosphodiesterase, amyloid β protein, protein phosphatase 2A, glycogen synthase kinase-3 beta, β-secretase, γ-secretase, α-secretase, serotonergic receptors, glutaminyl cyclase, tumor necrosis factor-α, γ-aminobutyric acid receptors, and mitochondria. All of these targets have been involved in the design of new potential drugs. An extensive number of these drugs have been studied in clinical trials. However, only galantamine, donepezil, and rivastigmine (ChEIs), memantine (NMDA antagonist), and aducanumab and lecanemab (selective anti-Aβ monoclonal antibodies) have been approved for AD treatment. Many drugs failed in the clinical trials to such an extent that questions have been posed about the significance of some of the aforementioned targets. On the contrary, the data of other drugs were promising and shed light on the significance of their targets for the development of new potent anti-alzheimer drugs.

Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Tumor targeted alpha particle therapy with an actinium-225 labelled antibody for carbonic anhydrase IX.

Selective antibody targeted delivery of α particle emitting actinium-225 to tumors has significant therapeutic potential. This work highlights the design and synthesis of a new bifunctional macrocyclic diazacrown ether chelator, H2MacropaSqOEt, that can be conjugated to antibodies and forms stable complexes with actinium-225. The macrocyclic diazacrown ether chelator incorporates a linker comprised of a short polyethylene glycol fragment and a squaramide ester that allows selective reaction with lysine residues on antibodies to form stable vinylogous amide linkages. This new H2MacropaSqOEt chelator was used to modify a monoclonal antibody, girentuximab (hG250), that binds to carbonic anhydrase IX, an enzyme that is overexpressed on the surface of cancers such as clear cell renal cell carcinoma. This new antibody conjugate (H2MacropaSq-hG250) had an average chelator to antibody ratio of 4 : 1 and retained high affinity for carbonic anhydrase IX. H2MacropaSq-hG250 was radiolabeled quantitatively with [225Ac]AcIII within one minute at room temperature with micromolar concentrations of antibody and the radioactive complex is stable in human serum for >7 days. Evaluation of [225Ac]Ac(MacropaSq-hG250) in a mouse xenograft model, that overexpresses carbonic anhydrase IX, demonstrated a highly significant therapeutic response. It is likely that H2MacropaSqOEt could be used to modify other antibodies providing a readily adaptable platform for other actinium-225 based therapeutics.

Selective antibody deficiency syndrome, response to vaccines, diagnosis, and treatment

Selective antibody deficiency syndrome (SAD), is a primary immunodeficiency in which immunoglobulin levels remain normal, but there is a reduced response to polysaccharide antigens after vaccination. SAD is recognized by the International Union of Immunology Societies as a primary immunodeficiency of unknown genetic cause. Patients with SAD are highly susceptible to severe respiratory tract infections with encapsulated bacteria. The infections found in SAD are similar to other antibody deficiencies; patients often present with recurrent upper and lower respiratory tract infections, otitis media, and sinusitis. The treatment of this pathology is based on preventing recurrent infections by known microorganisms, and in the case of serious infections, the use of intravenous or subcutaneous immunoglobulin.

Spesolimab for the Treatment of Generalized Pustular Psoriasis.

Generalized pustular psoriasis (GPP) is a rare but severe skin inflammatory disorder characterized by the eruption of widespread sterile neutrophilic pustules, often accompanied by systemic inflammation. Given its life-threatening potential, GPP requires prompt accurate diagnosis and effective treatment, but its rarity and relapsing-remitting nature pose challenges in performing large-scale randomized controlled clinical trials. Established international guidelines are currently lacking and management guidance often follows that for plaque psoriasis. However, while it can co-exist with plaque psoriasis and has traditionally been classified as a most severe form of psoriasis, GPP is now recognized as a distinct entity, with its own clinicopathological, autoinflammatory, immunologic and genetic features. Research conducted over the past decade revealed that an imbalance of interleukin (IL)-36 signaling favoring the proinflammatory activity is the central driver of the pathogenesis of GPP, thereby laying the groundwork for the development of targeted therapies for the disease. This article reviews the evidence thus far on spesolimab, a selective humanized antibody against the IL-36 receptor that was recently licensed in Europe and the United States for the treatment of GPP flares in adults. In phase II, randomized controlled clinical trials, spesolimab led to rapid and effective skin clearance in patients experiencing a GPP flare and demonstrated superiority to placebo in preventing flares for up to 48 weeks with maintenance treatment, with reassuring safety and tolerability profiles. Spesolimab is considered to be a first-in-class medication establishing itself as the standard of care for the treatment of GPP flares, thus changing the paradigm of the management of GPP to a new era of scientifically- and evidence-based targeted therapy for this distinctive disease.

Minimizing false positives for CTC identification

BackgroundCancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. ResultsThis study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. SignificanceThis study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.

Immunohistochemistry in pathology: A review

Immunohistochemistry (IHC) is an invaluable technique in pathology that allows for the localization and characterization of specific antigens within tissues using specific antibodies. Over the years, IHC has revolutionized the field of diagnostic pathology, providing critical information about the molecular and cellular composition of tissues. This comprehensive review article aims to provide an overview of the principles, methodologies, applications, and challenges of IHC in pathology. Immunohistochemistry (IHC) is a vital technique in pathology that allows for the detection and visualization of specific antigens within tissue samples. This article provides an overview of the principles, methodology, and applications of IHC in pathology. The principles of immunoreactivity, antibody selection, and detection methods are discussed, highlighting their importance in achieving accurate and reliable results. The methodology of IHC, including tissue preparation, antigen retrieval, and visualization techniques, is outlined to guide researchers and pathologists in implementing the technique effectively. The article also explores the role of antibodies in IHC, emphasizing the differences between monoclonal and polyclonal antibodies, as well as the significance of antibody validation and quality control. The applications of IHC in pathology are described, ranging from tumor classification and prognostic assessments to the detection of infectious agents and the characterization of autoimmune diseases. Furthermore, the article examines advancements and challenges in IHC, such as multiplex staining, digital pathology, quality control, and interpretation. Finally, emerging techniques, including the integration of next-generation sequencing and artificial intelligence, are discussed as future perspectives for IHC. Overall, this comprehensive overview demonstrates the indispensable role of IHC in pathology and its continuous evolution to meet the ever-expanding needs of diagnostic and research endeavors.