Bacterial coloration immunofluorescence strip for ultrasensitive rapid detection of bacterial antibodies and targeted antibody-secreting hybridomas.

The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering.

A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals.

Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.

Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19 / Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19

INTRODUÇÃO: O coronavírus da Síndrome Respiratória Aguda Grave 2 (abreviado para SARSCoV-2, do inglês Severe Acute Respiratory Syndrome Coronavirus 2), anteriormente conhecido como novo coronavírus (2019-nCoV), é um agente zoonótico recémemergente que surgiu em dezembro de 2019, em Wuhan, China, causando manifestações respiratórias, digestivas e sistêmicas, que se articulam no quadro clínico da doença denominada COVID-19 (do inglês Coronavirus Disease 2019). Ainda não há informações robustas sobre a história natural da doença, tampouco sobre as medidas de efetividade para o manejo clínico dos casos de infecção pelo COVID19, restando ainda muitos detalhes a serem esclarecidos. No entanto, sabe-se que o vírus tem alta transmissibilidade e provoca uma síndrome respiratória aguda que varia de casos leves ­ cerca de 80% ­ a casos muito graves com insuficiência respiratória - entre 5% e 10% dos casos ­, os quais requerem tratamento especializado em unidades de terapia intensiva (UTI). Sua letalidade varia, principalmente, conforme a faixa etária. TECNOLOGIA: Os testes de diagnóstico para a COVID-19 se destacaram na pandemia de coronavírus em andamento como uma ferramenta essencial para rastrear a propagação da doença. Uma ampla gama de testes diagnósticos para o SARS-CoV-2 está disponível comercialmente, alguns dos quais receberam autorizações para uso por várias agências reguladoras nacionais. Com as informações da sequência genética devidamente identificadas, os testes de diagnóstico baseados na detecção da sequência viral por reação em cadeia da polimerase com transcriptase reversa (RT-PCR) ou plataformas de sequenciamento logo se tornaram disponíveis. Isso permitiu a confirmação do diagnóstico e melhores estimativas da atividade da infecção, que vêm aumentando em velocidades alarmantes. Para a detecção mais sensível de SARS-CoV, MERS-CoV e SARS-CoV-2, recomendavam-se a coleta e o teste de amostras respiratórias superiores e inferiores. O diagnóstico de casos suspeitos era confirmado por testes de RNA com RT-PCR em tempo real ou sequenciamento de próxima geração. Foi demonstrado que o RNA viral poderia ser detectado a partir do swab nasal e faríngeo, lavagem broncoalveolar e plasma sanguíneo usando RT-PCR direcionado ao gene do vírus (5). O padrão-ouro para diagnóstico laboratorial da COVID-19 é a reação da transcriptase reversa, seguida de reação em cadeia da polimerase (RT-PCR) para amostras coletadas no trato respiratório superior ou inferior. OBJETIVO: O objetivo deste relatório é analisar a acurácia dos testes diagnósticos registrados na Agência Nacional de Vigilância Sanitária (ANVISA) até a presente data. METODOLOGIA: foi realizada uma busca por diagnósticos para COVID-19 com registros vigentes na ANVISA. Para tal, foram utilizados os termos "COVID 19", SARS e coronavírus no campo de consulta de registro de produtos para saúde no site da Agência (https://consultas.anvisa.gov.br/#/saude/). Os passos para acesso ao banco de dados de produtos diagnósticos na ANVISA são: 1) consulta produtos; 2) consulta a banco de dados; 3) produtos para a saúde e 4) pesquisa de produtos para a saúde registrados. CONCLUSÕES: A ANVISA já avaliou mais de 120 pedidos de registro de produtos para testagens relacionadas à COVID-19 desde o dia 18 de março. A maior parte das petições aguarda complementação de informações por parte das empresas e outras estão sendo analisadas com prioridade. O tempo médio para avaliação dos registros na ANVISA gira em torno de 15 dias. Atualmente, mais da metade dos registros concedidos diz respeito a testes rápidos para anticorpos. Até a presente data, foram registrados 64 testes para diagnóstico da COVID-19, sendo 15 deles moleculares. O teste de polymerase chain reaction em tempo real (RT-PCR) para identificação de SARS-CoV-2 é um teste de elevada sensibilidade e especificidade, ainda que os doentes com maior carga viral possam ter maior probabilidade de um teste positivo. Os testes moleculares baseados em RNA exigem instalações laboratoriais específicas com níveis restritos de biossegurança e técnica. A sensibilidade e especificidade dos testes sorológicos variaram entre os fabricantes. É importante destacar que uma baixa sensibilidade do teste diagnóstico pode resultar em uma maior probabilidade de detectar falsos-negativos, o que poderia interferir principalmente em casos de indivíduos assintomáticos. Em geral, a sensibilidade dos testes foi superior a 85% e a especificidade, superior a 94%. Os testes sorológicos medem a quantidade de dois anticorpos (IgG e IgM) que o organismo produz quando entra em contato com um invasor. Contudo, o desenvolvimento da resposta de um anticorpo à infecção pode ser dependente do hospedeiro e levar tempo. No caso de SARS-CoV-2, estudos iniciais sugerem que a maioria dos pacientes se converte entre 7 e 11 dias após a exposição ao vírus, embora alguns pacientes possam desenvolver anticorpos mais cedo. Devido a esse atraso natural, o teste de anticorpos pode não ser útil no cenário de uma doença aguda (11). Os testes de anticorpos para SARS-CoV-2 podem facilitar (i) o rastreamento de contatos (os testes baseados em RNA também podem ajudar); (ii) a vigilância sorológica nos níveis local, regional, estadual e nacional; e (iii) a identificação de quem já teve contato com o vírus e, portanto, pode (se houver imunidade protetora) ser imune (11,12). Alguns conjuntos de reagentes para testes sorológicos foram autorizados pela ANVISA em caráter emergencial devido à gravidade da situação e à necessidade de ampliar a testagem da população, mas a validação desses reagentes pelos laboratórios é fundamental, uma vez que poucos trabalhos conseguiram ser publicados até o momento. As aprovações estão de acordo com a Resolução da Diretoria Colegiada (RDC) 348/2020, que define os critérios e os procedimentos extraordinários e temporários para tratamento de petições de registro de medicamentos, produtos biológicos e produtos para diagnóstico in vitro, e mudança pós-registro de medicamentos e produtos biológicos em virtude da emergência de saúde pública internacional decorrente do novo coronavírus. Na RDC, para registro de testes diagnósticos, a ausência de qualquer estudo de desempenho ou restrição de dados deve ser justificada por motivações técnicas que permitam a avalição da confiabilidade dos resultados e da efetividade diagnóstica do produto. Os registros concedidos nas condições dessa Resolução terão a validade de um ano, exceto para situações em que a avaliação da estabilidade seja apresentada por comparação com produtos similares e os demais critérios descritos no Regulamento sejam atendidos. Nesse caso, poderão ter a concessão regular de validade de registro de produtos para saúde por um período de 10 anos. Em resumo, as duas categorias de testes para SARS-CoV-2 podem ser úteis nesse surto, pois, eventualmente, a coleta de múltiplas amostras, regiões e em tempos diferentes durante a evolução da doença pode ser necessária para o diagnóstico da COVID-19.

A microfabricated thickness shear mode electroacoustic resonator for the label-free detection of cardiac troponin in serum.

Portable, and automated biosensors for the detection of cardiac biomarkers are important for the early diagnosis of acute myocardial infarction. We demonstrate a label-free microfabricated thickness shear mode electroacoustic device based on a ZnO piezoelectric film for the assay of cardiac biomarkers. This device combines an immunologic sensitive element with the resonator working in biological liquids. Based on the gravimetric mechanism, the immunoreaction between the target antigens and antibodies was detected by monitoring the frequency change in real-time to determine the concentration of cardiac troponins I. The device exhibits a linear response from 0.04 to 2 ng/ml and a low detection limit of 20 pg/ml in serum containing cardiac troponin I. The clinical samples collected from healthy controls and cardiac patients were directly used without any additional pre-treatment. The cardiac troponin concentrations measured by the proposed device shows good linear correlation and consistency compared with standard laboratory methods. The results indicate that the device can to be a feasible tool for sensitive, rapid, and miniaturized point-of-care analytical systems.

Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.

Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.

Multiplex immunofluorescence staining and image analysis assay for diffuse large B cell lymphoma.

With the explosion of immuno-oncology and the approval of many immune checkpoint therapies by regulatory agencies in the last few years, understanding the tumor microenvironment (TME) in the context of patients' immune status has become essential. Among available immune profiling techniques, multiplex immunofluorescence (mIF) assays offer the unique advantage of preserving the architectural features of the tumor and revealing the spatial relationships between tumor cells and immune cells. A number of mIF and image analysis assays have been described for solid tumors but most are not sufficiently suitable in lymphoma, where the lack of clear tumor-stromal boundaries and high tumor density present significant challenges. Here we describe the development and optimization of a reliable workflow using Akoya Opal staining kits to label and analyze 6 markers per slide in diffuse large B-cell lymphoma (DLBCL) tissue sections. Five panels totaling 30 markers were developed to characterize infiltrating immune cells and relevant check-point proteins such as PD1, PD-L1, ICOS, SIRP-alpha and Lag3 on 70 DLBCL sections. Multiplexed sections were scanned using an Akoya multispectral scanner. An image analysis workflow using InForm and Matlab was developed to overcome challenges inherent to the DLBCL environment. Using the assays and workflows detailed here, we were able to quantify cell densities of subsets of infiltrating immune cells and observe their spatial patterns within the tumors. We highlight heterogeneous distribution of cytotoxic T cells across tumors with similar T cell density to underscores the importance of considering spatial context when studying the effects of immunological therapies in DLBCL.

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.

Analysis of primary cilia in renal tissue and cells.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.

Ultra-fast and automated immunohistofluorescent multistaining using a microfluidic tissue processor.

Multistaining of a tissue section targeting multiple markers allows to reveal complex interplays in a tumor environment. However, the resource-intensive and impractically long nature of iterative multiplexed immunostainings prohibits its practical implementation in daily routine, even when using work-flow automation systems. Here, we report a fully automated and ultra-fast multistaining using a microfluidic tissue processor (MTP) in as short as 20 minutes per marker, by immunofluorescent staining employing commercially available tyramide signal amplification polymer precipitation by horse-radish peroxidase (HRP) activation. The reported duration includes (i) 15 minutes for the entire fluidic exchange and reagent incubation necessary for the immunostaining and (ii) 5 minutes for the heat-induced removal of the applied antibodies. Using the automated MTP, we demonstrated a 4-plex automated multistaining with clinically relevant biomarkers within 84 minutes, showing perfect agreement with the state-of-the-art microwave treatment antibody removal. The presented HRP-based method is in principle extendable to multistaining by both tyramides accommodating higher number of fluorescent channels and multi-color chromogenic staining. We anticipate that our automated multi-staining with a turn-around time shorter than existing monoplex immunohistochemistry methods has the potential to enable multistaining in routine without disturbing the current laboratory workflow, opening perspectives for implementation of -omics approaches in tissue diagnostics.

A multichannel microchip containing 16 chambers packed with antibody-functionalized beads for immunofluorescence assay.

A multichannel chip containing 16 microchambers was developed for fast and sensitive immunoassays. In each chamber, antibody-functionalized nonmagnetic beads were applied as the solid phase to capture target antigens. Four types of IgGs (human, rabbit, chicken, and mouse) could be detected simultaneously by our combining this microchip with a sandwich immunoassay technique. A three-layer chip structure was investigated for integration of multiple processes, including washing, immune reaction, and detection, in one microchip. Moreover, the proposed chip design could improve batch-to-batch repeatability and avoid interferences between different channels without the preparation of complex microvalves. The total operation time of this system was less than 30 min, with a desirable detection limit of 0.2 pg/mL. The results indicate that the microfluidic platform is promising for the immunoassay of multiple clinical biomarkers. Graphical abstract.

Immunostaining of Skeletal Tissues.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

Immuno-gold silver staining assays on capillary-driven microfluidics for the detection of malaria antigens.

Accurate and affordable rapid diagnostic tests (RDTs) are indispensable but often lacking for many infectious diseases. Specifically, there is a lack of highly sensitive malaria RDTs that can detect low antigen concentration at the onset of infection. Here, we present a strategy to improve the sensitivity of malaria RDTs by using capillary-driven microfluidic chips and combining sandwich immunoassays with electroless silver staining. We used 5 µm fluorescent beads functionalized with capture antibodies (cAbs). These beads are self-assembled by capillary action in recessed "bead lanes", which cross the main flow path of chips microfabricated in Si and SU-8. The binding of analytes to detection antibodies (dAbs) and secondary antibodies (2ndAbs) conjugated to gold nanoparticles (NPs) allows the formation of a silver film on the beads. Such silver film masks the fluorescent core of the bead inversely proportional to the concentration of antigen in a sample. We illustrate this method using the recombinant malaria antigen Plasmodium falciparum histidine-rich-protein 2 (rPfHRP2) spiked in human serum. This antigen was a recombinant HRP2 protein expressed in Escherichia coli, which is also the standard reference material. The limit of detection (LOD) of our immunoassay was found to be less than 6 ng mL-1 of rPfHRP2 within 20 min, which is approaching the desired sensitivity needed in the Target Product Profile (TPP) for malaria elimination settings. The concept presented here is flexible and may also be utilized for implementing fluorescence immunoassays for the parallel detection of biomarkers on capillary-driven microfluidic chips.

Tape mount immunostaining: a versatile method for immunofluorescence analysis of fungi.

AIM: Immunofluorescence microscopy is a powerful technique to detect surface antigens and study their distribution. Analysis of fungi is often hampered by their weak adherence to glass. We therefore established a novel immunofluorescence staining method to overcome this problem. MATERIALS & METHODS: Fungal material from colonies is bound to adhesive tape and stained with antibodies. RESULTS: The obtained samples had very good optical quality, showing low unspecific background staining and allowing analysis by confocal laser scanning microscopy. We have exemplified applying the new method to study the distribution of galactomannan on conidiophores of Aspergillus fumigatus and of ß-glucans on Malassezia pachydermatis. CONCLUSION: Tape mount immunostaining facilitates analysis of fungal surface molecules and provides a base for expeditious diagnostic procedures.

Double Immunohistochemistry and Digital Image Analysis.

Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

Characterization of the "Autophagic Flux" in Prostate Cancer Tissue Biopsies by LC3A/LAMP2a Immunofluorescence and Confocal Microscopy.

A method is described for the immunostaining of human prostate cancer biopsies for the autophagic marker LC3 and the lysosomal protein LAMP2A. Using this combination we can provide some information on autophagic flux, and specific patterns of staining have been recognized for normal prostate tissue and prostatic carcinomas.

Methods for Evaluation of Ovarian Granulosa Cells with Exposure to Nanoparticles.

Increasing evidence demonstrates that a substantial number of chemicals including nanoparticles have potential toxic effects on human and wildlife reproductive health. Ovarian granulosa cells (GCs), the functional unit of the ovary, play a pivotal role in the regulation of ovarian reproductive health in females. They are considered to be the main target by the chemicals at all stages of their development. It has been reported that nanoparticles induced toxicity of GCs by interfering with cell cycle. Some basic methods for evaluation of GCs with exposure to nanoparticles are described in this chapter including isolation of GCs, determination of apoptosis by immunofluorescence, and measurement of progesterone level by ELISA.

Determination of Phosphorylated Histone H2AX in Nanoparticle-Induced Genotoxic Studies.

DNA double-strand breaks (DSBs), one of the most severe lesions of DNA damage triggered by various genotoxic insults, can lead to chromosome change, genomic instability, and even tumorigenesis if not repaired efficiently. In response to DNA damage, histone H2AX molecules are rapidly phosphorylated at serine 139 near the site of DNA DSBs and form γ-H2AX foci. As an early important cellular event linked to DNA damage and repair, γ-H2AX is a highly sensitive biomarker for "monitoring" DNA damage and consequently is a useful tool in genetic toxicology screen. We and other researchers have used γ-H2AX as a marker to assess the potential genotoxic effects of some nanoparticles in vitro and in vivo. In this chapter, we describe several useful methods for γ-H2AX detection, which can be used to evaluate the potential genotoxic effects of nanoparticles.

Assays on DNA Damage and Repair in CLL.

Assays that measure DNA damage and repair are critical in evaluating the extent to which therapeutic agents damage DNA and in identifying whether DNA repair can limit the toxicity of chemotherapy. The COMET assays described in this guide should help readers evaluate single and double-strand breaks cause by chemotherapeutic agents and also monitor the ability of the cells to repair such damage. The EJDR assay described is a valuable tool to assess the ability of drugs and DNA repair proteins to modulate DNA repair capacity. Finally, the immunofluorescence assay described should allow accurate assessments of DNA damage and the kinetics of repair as measured by Ɣ-H2AX foci. This procedure can also be used to mechanistically investigate the recruitment of specific DNA damage and repair proteins in CLL cells.