Optimized immunofluorescence staining protocol for imaging germinal centers in secondary lymphoid tissues of vaccinated mice.

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

Multiphoton Deep-Tissue Imaging of Micrometastases and Disseminated Cancer Cells Using Conjugates of Quantum Dots and Single-Domain Antibodies.

Early detection of malignant tumors, micrometastases, and disseminated tumor cells is one of the effective way of fighting cancer. Among the many existing imaging methods like computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), optical imaging with fluorescent probes is one of the most promising alternatives because it is fast, inexpensive, safe, sensitive, and specific. However, traditional fluorescent probes, based on organic fluorescent dyes, suffer from the low signal-to-noise ratio. Furthermore, conventional organic fluorescent dyes are unsuitable for deep tissue imaging because of the strong visible light absorption by biological tissues. The use of fluorescent semiconductor nanocrystals, or quantum dots (QDs), may overcome this limitation due to their large multiphoton cross section, which ensures efficient imaging of thick tissue sections inaccessible with conventional fluorescent probes. Moreover, the lower photobleaching and higher brightness of fluorescence signals from QDs ensures a much better discrimination of positive signals from the background. The use of fluorescent nanoprobes based on QDs conjugated to uniformly oriented high-affinity single-domain antibodies (sdAbs) may significantly increase the sensitivity and specificity due to better recognition of analytes and deeper penetration into tissues due to small size of such nanoprobes.Here, we describe a protocol for the fabrication of nanoprobes based on sdAbs and QDs, preparation of experimental xenograft mouse models for quality control, and multiphoton imaging of deep-tissue solid tumors, micrometastases, and disseminated tumor cells.

Spatial Analysis and Clinical Significance of HLA Class-I and Class-II Subunit Expression in Non-Small Cell Lung Cancer.

PURPOSE: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of ß2 microglobulin (ß2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II ß chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. RESULTS: Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. ß2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of ß2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific ß2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8+ T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival. CONCLUSIONS: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.

Performance evaluation of Automated Fluorescent Immunoassay System ROTA and NORO for detection of rotavirus and norovirus: A comparative study of assay performance with RIDASCREEN® Rotavirus and Norovirus.

BACKGROUND: The Automated Fluorescent Immunoassay System ROTA (AFIAS-Rota) and NORO (AFIAS-Noro) assays (Boditech Med Inc.) are newly developed diagnostic tests for rotavirus and norovirus infections. METHODS: Performance of AFIAS-Rota/Noro assays was evaluated in comparison with RIDASCREEN® Rotavirus and Norovirus ELISA kits (R-Biopharm) using clinical stool samples submitted from November 2018 to January 2019. Multiplex real-time reverse transcription-polymerase chain reaction was used as reference method. RESULTS: A total of 256 clinical specimens were analyzed. AFIAS-Rota and RIDASCREEN Rotavirus had almost perfect agreement (Kappa value = 0.95), and substantial agreement was observed between AFIAS-Noro and RIDASCREEN Norovirus (Kappa value = 0.80). For detection of rotavirus, AFIAS and RIDASCREEN assays showed satisfactory diagnostic sensitivity (100% and 97.8%, respectively) and specificity (99.5% and 99.1%). For detection of norovirus, the RIDASCREEN assay showed significantly higher sensitivity than the AFIAS-Noro (86.0% and 66.0%, respectively; P = .002). Analytic specificity of AFIAS-Rota/Noro assays showed no cross-reactivity against any other bacteria (14 strains) or viruses (2 strains). Hands-on time (6 minutes) and turnaround time (26 minutes) required to perform AFIAS assays were much shorter than those required for RIDASCREEN assays (20 and 150 minutes, respectively). CONCLUSION: The AFIAS-Rota/Noro assays showed overall excellent agreement with the RIDASCREEN assays. Although the AFIAS-Noro assay exhibited lower sensitivity than the RIDASCREEN Norovirus assay for detection of norovirus, the AFIAS-Rota/Noro assays could be useful as a rapid initial screening test in clinical laboratories due to its convenience and rapid turnaround time.

An improved and simplified protocol to combine Golgi-Cox staining with immunofluorescence and transmission electron microscopy techniques.

Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.

Long-Term Variability in Immunofluorescence Titer of Antibodies to Nuclear Antigens Observed in Clinical Laboratory Proficiency Testing Surveys.

CONTEXT.­: Presence of antibodies to nuclear antigens (ANAs) above a threshold titer is an important diagnostic feature of several autoimmune diseases, yet titers reported vary between laboratories. Proficiency survey results can help clarify factors contributing to the variability. OBJECTIVE.­: To determine the contribution of HEp-2 ANA kits from different manufacturers to the variation in titers, and assess whether the differences between kits are consistent over the long term. DESIGN.­: HEp-2 ANA titers reported by laboratories participating in the external quality assessment proficiency testing surveys conducted by the College of American Pathologists between 2008 and 2018 were analyzed. The ANA titers reported for each specimen were ranked according to the kits being used by testing laboratories, and the statistical significance of the differences was determined. RESULTS.­: The ANA titer results were strongly influenced by the HEp-2 ANA kit used (P < .001). During the 11 years studied, the rank order of the ANA titer for each kit relative to the other kits was remarkably consistent. The rank of ANA titer for individual ANA patterns observed for each kit was similar to the overall rank of that kit. CONCLUSIONS.­: Variability in ANA titers was strongly associated with the kits used, and the differences between kits were quite consistent during the 11 years studied. Because the variability is not random, it has the potential to be managed by harmonizing kits, which could lead to improved consistency in reporting ANA titers.

Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice.

BACKGROUND: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. METHODS: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. RESULTS: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. CONCLUSIONS: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.

Advances in tissue-based imaging: impact on oncology research and clinical practice.

INTRODUCTION: Tissue-based imaging has emerged as a critical tool in translational cancer research and is rapidly gaining traction within a clinical context. Significant progress has been made in the digital pathology arena, particularly in respect of brightfield and fluorescent imaging. Critically, the cellular context of molecular alterations occurring at DNA, RNA, or protein level within tumor tissue is now being more fully appreciated. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumor microenvironment, including the potential interplay between various cell types. AREAS COVERED: This review summarizes the recent developments within the field of tissue-based imaging, centering on the application of these approaches in oncology research and clinical practice. EXPERT OPINION: Significant advances have been made in digital pathology during the last 10 years. These include the use of quantitative image analysis algorithms, predictive artificial intelligence (AI) on large datasets of H&E images, and quantification of fluorescence multiplexed tissue imaging data. We believe that new methodologies that can integrate AI-derived histologic data with omic data, together with other forms of imaging data (such as radiologic image data), will enhance our ability to deliver better diagnostics and treatment decisions to the cancer patient.

An image dataset related to automated macrophage detection in immunostained lymphoma tissue samples.

BACKGROUND: We present an image dataset related to automated segmentation and counting of macrophages in diffuse large B-cell lymphoma (DLBCL) tissue sections. For the classification of DLBCL subtypes, as well as for providing a prognosis of the clinical outcome, the analysis of the tumor microenvironment and, particularly, of the different types and functions of tumor-associated macrophages is indispensable. Until now, however, most information about macrophages has been obtained either in a completely indirect way by gene expression profiling or by manual counts in immunohistochemically (IHC) fluorescence-stained tissue samples while automated recognition of single IHC stained macrophages remains a difficult task. In an accompanying publication, a reliable approach to this problem has been established, and a large set of related images has been generated and analyzed. RESULTS: Provided image data comprise (i) fluorescence microscopy images of 44 multiple immunohistostained DLBCL tumor subregions, captured at 4 channels corresponding to CD14, CD163, Pax5, and DAPI; (ii) "cartoon-like" total variation-filtered versions of these images, generated by Rudin-Osher-Fatemi denoising; (iii) an automatically generated mask of the evaluation subregion, based on information from the DAPI channel; and (iv) automatically generated segmentation masks for macrophages (using information from CD14 and CD163 channels), B-cells (using information from Pax5 channel), and all cell nuclei (using information from DAPI channel). CONCLUSIONS: A large set of IHC stained DLBCL specimens is provided together with segmentation masks for different cell populations generated by a reference method for automated image analysis, thus featuring considerable reuse potential.

Advances in quantitative immunohistochemistry and their contribution to breast cancer.

Introduction: Automated image analysis provides an objective, quantitative, and reproducible method of measurement of biomarkers. Image quantification is particularly well suited for the analysis of tissue microarrays which has played a major pivotal role in the rapid assessment of molecular biomarkers. Data acquired from grinding up bulk tissue samples miss spatial information regarding cellular localization; therefore, methods that allow for spatial cell phenotyping at high resolution have proven to be valuable in many biomarker discovery assays. Here, we focus our attention on breast cancer as an example of a tumor type that has benefited from quantitative biomarker studies using tissue microarray format.Areas covered: The history of immunofluorescence and immunohistochemistry and the current status of these techniques, including multiplexing technologies (spectral and non-spectral) and image analysis software will be addressed. Finally, we will turn our attention to studies that have provided proof-of-principle evidence that have been impacted from the use of these techniques.Expert opinion: Assessment of prognostic and predictive biomarkers on tissue sections and TMA using Quantitative immunohistochemistry is an important advancement in the investigation of biologic markers. The challenges in standardization of quantitative technologies for accurate assessment are required for adoption into routine clinical practice.

International multicenter examination of MOG antibody assays.

OBJECTIVE: To compare the reproducibility of 11 antibody assays for immunoglobulin (Ig) G and IgM myelin oligodendrocyte glycoprotein antibodies (MOG-IgG and MOG-IgM) from 5 international centers. METHODS: The following samples were analyzed: MOG-IgG clearly positive sera (n = 39), MOG-IgG low positive sera (n = 39), borderline negative sera (n = 13), clearly negative sera (n = 40), and healthy blood donors (n = 30). As technical controls, 18 replicates (9 MOG-IgG positive and 9 negative) were included. All samples and controls were recoded, aliquoted, and distributed to the 5 testing centers, which performed the following antibody assays: 5 live and 1 fixed immunofluorescence cell-based assays (CBA-IF, 5 MOG-IgG, and 1 MOG-IgM), 3 live flow cytometry cell-based assays (CBA-FACS, all MOG-IgG), and 2 ELISAs (both MOG-IgG). RESULTS: We found excellent agreement (96%) between the live CBAs for MOG-IgG for samples previously identified as clearly positive or negative from 4 different national testing centers. The agreement was lower with fixed CBA-IF (90%), and the ELISA showed no concordance with CBAs for detection of human MOG-IgG. All CBAs showed excellent interassay reproducibility. The agreement of MOG-IgG CBAs for borderline negative (77%) and particularly low positive (33%) samples was less good. Finally, most samples from healthy blood donors (97%) were negative for MOG-IgG in all CBAs. CONCLUSIONS: Live MOG-IgG CBAs showed excellent agreement for high positive and negative samples at 3 international testing centers. Low positive samples were more frequently discordant than in a similar comparison of aquaporin-4 antibody assays. Further research is needed to improve international standardization for clinical care.

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

Utilizing supervised machine learning to identify microglia and astrocytes in situ: implications for large-scale image analysis and quantification.

BACKGROUND: The evaluation of histological tissue samples plays a crucial role in deciphering preclinical disease and injury mechanisms. High-resolution images can be obtained quickly however data acquisition are often bottlenecked by manual analysis methodologies. NEW METHOD: We describe and validate a pipeline for a novel machine learning-based analytical method, using the Opera High-Content Screening system and Harmony software, allowing for detailed image analysis of cellular markers in histological samples. RESULTS: To validate the machine learning pipeline, analyses of single proteins in mouse brain sections were utilized. To demonstrate adaptability of the pipeline for multiple cell types and epitopes, the percent brain coverage of microglial cells, identified by ionized calcium binding adaptors molecule 1 (Iba1), and of astrocytes, by glial fibrillary acidic protein (GFAP) demonstrated no significant differences between automated and manual analyses protocols. Further to examine the robustness of this protocol for multiple proteins simultaneously labeling of rat brain sections were utilized; co-localization of astrocytic endfeet on blood vessels, using aquaporin-4 and tomato lectin respectively, were efficiently identified and quantified by the novel pipeline and were not significantly different between the two analyses protocols. Comparison with Existing Methods: The automated platform maintained the sensitivity and accuracy of manual analysis, while accomplishing the analyses in 1/200th of the time. CONCLUSIONS: We demonstrate the benefits and potential of adapting an automated high-throughput machine-learning analytical approach for the analysis ofin situ tissue samples, show effectiveness across different animal models, while reducing analysis time and increasing productivity.

[Standardization of component manufacturing conditions and setting up the immunofluorescence reaction on the example of syphilis laboratory diagnostics (literature review).]

The use of technology for preparing components for immunofluorescence reaction (RIF) in a number of clinical diagnostic laboratories (CDL) in accordance with the recommendations of the Guidelines introduced by order of the RF Ministry of Health No 87 dated March 26, 2001 does not guarantee the required standardization of RIF preparation and conduct conditions. The latter is achieved by using the appropriate industrial production reagent kits in the СDL practice.

Evaluation of the Sofia S. pneumoniae FIA for Detection of Pneumococcal Antigen in Patients with Bloodstream Infection.

The usefulness of pneumococcal urinary antigen tests (UATs) in severe pneumococcal infection relies heavily on the performance in bacteremic patients. Fluorescence technology and automatic reading of test results may improve UAT performance. We evaluated the automatically read Sofia S. pneumoniae FIA for diagnosing pneumococcal bloodstream infection (BSI) in hospitalized adult patients. First, the Sofia FIA was evaluated on 97 patients with pneumococcal (n = 47) and nonpneumococcal (n = 50) BSI and compared with results by the visually read BinaxNOW S. pneumoniae immunochromatographic test (ICT) and ImmuView S. pneumoniae and Legionella pneumophila ICT. In four cases (4.1%), the Sofia FIA showed invalid test results, three of which showed invalid results by the ImmuView ICT previously. Based on 93 valid cases, the Sofia FIA showed similar sensitivity (for both comparisons: 68% versus 62%; P = 0.45) and specificity (for both comparisons: 91% versus 93%; P = 1.00) as the visually read UATs. Second, the Sofia FIA was prospectively evaluated on 82 consecutive nonfrozen urine samples, detecting pneumococcal antigen in 10 of 14 (sensitivity, 71%) pneumococcal BSI patients, similarly to the visually and automatically read BinaxNOW ICT (both 12 of 14; sensitivity, 86%; P = 0.50). Of five nonpneumococcal BSI cases, the Sofia FIA showed an invalid test result in one case, but no positive UAT results were obtained. Thus, the sensitivity and specificity of the Sofia FIA were similar to the performance rates of other UATs in patients with BSI, but invalid test results are of concern for the usefulness in pneumococcal BSI.

Unsupervised quantification of tissue immunofluorescence in animal models of multiple sclerosis - Instructions for use.

BACKGROUND: In the analysis of animal models of CNS diseases such as experimental autoimmune encephalomyelitis (EAE), immunostaining and histopathology are important readouts. However, the complex morphological features of a tissue staining are often reduced to a single measure which relies on tedious manual planimetry. Furthermore, the measure itself and co-variables such as the region being analysed are chosen in a human decision-making process, which introduces bias. NEW METHOD: First aim of the present study is to provide an open-source workflow for the high-throughput, unsupervised quantification of different stainings in the spinal cord. We evaluate different EAE models, spinal cord regions and different time points of disease. By applying random forest classification, we compare different measures. RESULTS: Exemplified for glial reactivity, we show that measures and variables interact and that their values are non-normally distributed, hampering the common use of parametric tests. Furthermore, we demonstrate that one-dimensional measures are insufficient descriptors for immunofluorescence data in EAE and thus need to be considered as partly invalid. COMPARISON WITH EXISTING METHODS: We show in a systematic analysis of EAE studies that currently published immunohistological outcomes are highly incompatible regarding methodology and statistics. Furthermore, they lack the report of important information necessary for reproducibility and do not use unsupervised automatic analysis. CONCLUSIONS: Our results discover relevant caveats in the currently used methods of immunofluorescence analysis. The provided step-by-step instructions and open-source code are intended to serve as a framework for sensitive, unbiased immunofluorescence analysis of tissue sections in translational research.

Prospective evaluation of diagnostic tools for respiratory viruses in children and adults.

AIM: To compare the performances of molecular and non-molecular tests to diagnose respiratory viral infections and to evaluate the pros and contras of each technique. METHODS: Two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (FilmArray Respiratory Panel, Clart Pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. FINDINGS: Molecular techniques permitted the recovery of up to 50% more respiratory pathogens in comparison to non-molecular methods. FilmArray detected at least 30% more pathogens than Clart Pneumovir which could be explained by the differences in their technical designs. The turnaround time under 2 hours for the FilmArray permitted delivery of results when patients were still in the emergency room.

Antinuclear antibodies detection: A comparative study between automated recognition and conventional visual interpretation.

BACKGROUND: The indirect immunofluorescence assay (IIFA) for the detection of antinuclear antibodies (ANA) was firstly described in 1958 and is still considered the reference method for ANA screening. Currently, an automated processing and recognition system for standardized and efficient ANA interpretation by human epithelial (HEp-2) cell-based immunofluorescence (IIF; EUROPattern Suite, Euroimmun) is available in China. METHODS: In this study, the performance of this novel system for positive/negative classification, pattern recognition (including homogenous, speckled, nucleolar, nuclear dots, cytoplasmic, and centromeres patterns) and titers evaluation was evaluated by comparing to visual interpretation. RESULTS: Referring to the total of 3681 collected samples, there was an agreement of 98.7% (κ = 0.973) between the visual and automated examination regarding positive/negative discrimination. In sera with single pattern, correct pattern recognition was observed in 94.6% of the samples. The efficiency of automated recognition for single pattern varied for the different patterns. The automatically determined patterns were correct and complete in 1071 of 1620 cases and correct and meaningful but not complete ("main pattern") in another 405 cases, enabling main pattern recognition in 91.1% of all cases. Referring to the titers evaluation, the results within the next titer were considered to be consistent. In 1603 positive sera both by visual and automated evaluation, titers of 1514 sample were consistent, accounting for 94.4%. CONCLUSION: Attributed to the performance characteristics, EUROPattern system is suitable for clinical use as its high degree of automation and result reliability, and may help clinical laboratories to standardize of IIF evaluation.

Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre- and post-photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high-sensitivity LX-200 system using photobleached magnetic beads, human interleukin-8 is detected with a threefold improvement in detection limit and signal-to-noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence-based assays.