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BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

IntroductionGastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models.MethodsMKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 μg) response curves were performed in subcutaneous models. Orthotopic models received 50 μg of M5A-IR800 or 50 μg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System.ResultsIn subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors.ConclusionsHumanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection.

Abstract Spatial proteomics is a multidimensional technique that studies the spatial distribution and function of proteins within cells or tissues across both spatial and temporal dimensions. This field multidimensionally reveals the complex structure of the human proteome, including the characteristics of protein spatial distribution, dynamic protein translocation, and protein interaction networks. Recently, as a crucial method for studying protein spatial localization, spatial proteomics has been applied in the clinical investigation of various diseases. This review summarizes the fundamental concepts and characteristics of tissue-level spatial proteomics, its research progress in common human diseases such as cancer, neurological disorders, cardiovascular diseases, autoimmune diseases, and anticipates its future development trends. The aim is to highlight the significant impact of spatial proteomics on understanding disease pathogenesis, advancing diagnostic methods, and developing potential therapeutic targets in clinical research.

Background/Objectives: Pancreatic cancer is the third leading cause of death related to cancer. The only possible cure presently is complete surgical resection; however, this is limited by difficulty in clearly defining tumor margins. Enhancement of the visualization of pancreatic ductal adenocarcinoma (PDAC) tumor margins using near-infrared dye-conjugated tumor-specific antibodies was pioneered by using anti-CEA, anti-CA19.9, and anti-MUC5AC in orthotopic mouse models of pancreatic cancer. Recently, an antibody to Mucin 4 (MUC4) conjugated to a fluorescent probe has shown promise in targeting colon tumors in orthotopic mouse models. Methods: In the present study, we targeted pancreatic cancer using an anti-MUC4 antibody conjugated to IRDye800 (anti-MUC4-IR800) in orthotopic mouse models. Two pancreatic cancer human cell lines were used, SW1990 and CD18/HPAF. Results: Anti-MUC4-IR800 targeted the two pancreatic cancer cell line tumors in orthotopic mouse models with high tumor-to-pancreas ratios and high tumor-to-liver ratios, with greater targeting seen in SW1990. Conclusions: The present results suggest anti-MUC4-IR800's potential to be used in fluorescence-guided surgical resection of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Spatial proteomics is a multidimensional technique that studies the spatial distribution and function of proteins within cells or tissues across both spatial and temporal dimensions. This field multidimensionally reveals the complex structure of the human proteome, including the characteristics of protein spatial distribution, dynamic protein translocation, and protein interaction networks. Recently, as a crucial method for studying protein spatial localization, spatial proteomics has been applied in the clinical investigation of various diseases. This review summarizes the fundamental concepts and characteristics of tissue-level spatial proteomics, its research progress in common human diseases such as cancer, neurological disorders, cardiovascular diseases, autoimmune diseases, and anticipates its future development trends. The aim is to highlight the significant impact of spatial proteomics on understanding disease pathogenesis, advancing diagnostic methods, and developing potential therapeutic targets in clinical research. [ABSTRACT FROM AUTHOR]

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

Gastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models. MKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 μg) response curves were performed in subcutaneous models. Orthotopic models received 50 μg of M5A-IR800 or 50 μg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. In subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors. Humanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. Key points: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates [ABSTRACT FROM AUTHOR]

Background/aimFluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models.Materials and methodsHumanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated.ResultsIntra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76).ConclusionAnti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

The canine distemper virus (CDV), which infects dogs and a broad range of animal species, remains a serious concern in Türkiye and across the world. The current study shows that CDV can be detected and isolated rapidly and specifically in naturally infected dogs. Whole blood, nasal swab, ocular swab, rectal swab, and urine samples from 50 stray dogs were used in the study (n = 250). The presence of the CDV genome was confirmed in 105 (42%) samples using one-Step real-time RT-PCR. In total, 39 dogs were diagnosed with CDV infection based on the detection of cytopathic effects in MDCK, which was verified by the fluorescent antibody technique. A total of 12 one-Step real-time RT-PCR negative samples, consisting of 4 rectal swabs and 8 urine samples, were found to be positive by virus isolation. Blood, nasal swab, ocular swab (P<0.01, r = 1), rectal swab (P<0.01, r = 0.844), and urine samples (P<0.01, r = 0.697) all showed positive correlations in the tests for viral genome detection and virus isolation. CPE levels of high 37 (31.62%), medium 26 (22.23%) and low 54 (46.15%) were detected in a total of 117 (46.8%) samples with viral growth in cell culture. The highest CPE levels detected by FAT were for rectal swab and urine samples. In conclusion, the one-step real-time RTPCR method on rectal swab samples proved to be a very sensitive method for the rapid and reliable CDV detection. Besides, non-modified MDCK can be used to isolate CDV from naturally infected dogs. [ABSTRACT FROM AUTHOR]

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry. [ABSTRACT FROM AUTHOR]

Molecular endoscopy in inflammatory bowel disease (IBD) has made the translation from preclinical studies to clinical trials. The so far performed in vivo studies, using fluorescent antibodies, have addressed areas of heightened clinical interest with unmet needs. These include the distribution of targeted therapies within the mucosa, which could elucidate the most fitting dosing for the individual patient, the mode of action of currently used treatments, and subsequently also the prediction of therapeutic response. Altogether, molecular endoscopy might enable us to base individualized therapeutic decisions on preceded in vivo molecular analysis to optimize treatment in IBD., Competing Interests: Disclosure R. Atreya has served as a speaker, or consultant, or received research grants from AbbVie, Abivax, AstraZeneca, Bristol-Myers Squibb, Celltrion Healthcare, Galapagos, Johnson&Johnson, Lilly, MSD, Pfizer, and Takeda Pharma. DFG-SFB/TRR241 Project No. C02 and IBDome, CRU5024 B04 are funded by the German Research Council (DFG). Further supported by the FIBROTARGET project. The FIBROTARGET project has received funding from the European Union’s Horizon Europe Research & Innovation program under grant agreement no. 101080523. Funded by the European Union. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them. T. Rath has received speaker fees from Olympus Medical, PENTAX Medical, Mauna Kea Technologies, Medtronic, Takeda, Galapagos, Falk, Janssen, AbbVie, Repha, Medical Tribune, and Lilly. M F. Neurath has served as advisor or speaker for Pentax, Roche Pharma, Takeda Pharma, Pfizer, MSD, PPM, Janssen, Gilead, Dr Falk Pharma, Boehringer Ingelheim, Amgen, and AbbVie., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of pancreatic cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm or 650 nm fluorophores. Western blotting confirmed the expression of IGF-1R in Panc-1, BxPC3, and MIAPaCa-2 human pancreatic cancer cell lines. Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of the pancreatic cancer cells. Subcutaneous Panc-1, BxPC-3, and MIA PaCa-2 tumors became fluorescent after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted BxPC-3 tumors became fluorescent with the conjugated IGF-1R antibodies, and were easily visible with intravital imaging. Gross and microscopic ex vivo imaging of resected pancreatic tumor and normal pancreas confirmed that fluorescence indeed came from the membrane of cancer cells, and it was stronger from the tumor than the normal tissue. The present study demonstrates that fluorophore-conjugated IGF-1R antibodies can visualize pancreatic cancer and it can be used with various imaging devices such as endoscopy and laparoscopy for diagnosis and fluorescence-guided surgery.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.

Summary: Foodborne pathogens such as Salmonella enterica serovar Typhimurium, which cause self‐limiting gastroenteritis, are important in food safety and public health. Detection of Salmonella in complex food matrices is required for rapid and effective monitoring of contaminated food products. In this study, we collected Salmonella Typhimurium in chicken using immunomagnetic separation after formaldehyde treatment. Collected Salmonella Typhimurium cells were then quantified microfluidic device or fluorescence microscopy. The recovery ratio obtained with immunomagnetic separation after formaldehyde treatment was 77 (±5)% for the microscope count and 63 (±9)% for the microfluidic count. According to the fluorescence microscope images, the food matrix was effectively removed from the collected cell suspension. The counts of Salmonella Typhimurium cells obtained by the microfluidic device and fluorescence microscope were closely correlated (R2 = 0.993) with a range of 3.3 (±0.5) × 103 to 1.2 (±0.5) × 106 cells g−1. Our method provides quantitative detection of Salmonella Typhimurium within 3–4 h and could be used to efficiently evaluate microbial risk of quality control of foodstuffs and prevention of foodborne disease. [ABSTRACT FROM AUTHOR]

Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact. [ABSTRACT FROM AUTHOR]

The development of a simple and rapid technique for detecting programmed cell death-ligand 1 (PDL1) is crucial for cancer diagnosis and therapeutic monitoring. In this study, a novel Quenchbody (Q-body) was developed for rapid and high-throughput one-pot detection of PDL1. Specifically, a new labeling method using both Cystagging and Sortagging was employed to conjugate two dyes of different colors to the anti-PDL1 antibody fragment. The Q-body represented a PDL1 concentration-dependent fluorescence response, and the signal of the hetero-dye-labeled Q-body was enhanced compared with that of a single-dye-labeled Q-body. The anti-PDL1 Q-body generated herein can be used for various applications as a potential diagnostic probe, including screening PDL1 overexpressing cancer cells to facilitate personalized cancer therapy. • A novel Quenchbody was generated for one-pot detection of PDL1. • Anti-PDL1 scFv was labeled with two different dye colors to enhance the antibody response. • A procedure for expressing scFv using E. coli and transforming it into a Q-body was first pioneered in this study. [ABSTRACT FROM AUTHOR]

The paired detection of the transcriptome and proteome at single-cell resolution provides exquisite insight to immune mechanisms in health and disease. Here, we describe a detailed protocol wherein we combine cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), a technique utilizing antibody-derived tags (ADTs) to profile mRNA and proteins simultaneously via sequencing, with fluorescence-activated cell sorting to enrich cell populations. Our protocol provides step-by-step guidance on co-staining cells with both fluorescent antibodies and ADTs simultaneously, instructions on cell sorting and an overview of the single-cell capture workflow using the BD Rhapsody™ system. This method is useful for in-depth single-cell characterization on sorted rare cell populations., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

[Objectives] The objective of this project is to determined whether the recombinant fluorescent antibody expressed in Escherichia coli can be used to detect Classical swine fever virus(CSFV) in direct immune fluorescence assay. [Methods] The gene VHH-EGFP synthesized according to condon usage of E.coli was cloned into pET32a (+) and then transformed into E.coli BL21(DE3). The recombinant cells were cultured in LB (containing 50 μg·mL-1 of ampicillin) and incubated in 37 °C until A600=0.6-0.8, then the target gene was induced with 0.4 mmol·L-1 IPTG for 24 h at 15 °C. The induced bacterial cell pellet was collected and cracked, the interest protein was identified by 120 g·L-1 SDS-PAGE and Western blot, after confirmation, the expressed target protein was purified with Ni-NTA His·Bind Resin and detemined by BCA protein assay kit, and then it was incubated with cells infected with CSFV, PCV2, PRV, PPV to analyze its specificity and sensitivity. [Results] SDS-PAGE and Western-blot indicated that the recombinant fluorescent antibody was successfully expressed in E.coli BL21(DE3). Although it was mainly expressed as inclusion body form, the soluble protein could be purified and concentrated by nickel column. The PK15 cells infected with CSFV showed numerous obvious green fluorescences points after it was incubated with recombinant antibody. However, there was no or only few nonspecific fluorescence when the recombinant protein incubates with cells infected with PCV2, PRV, PPV. Compared with the IFA based on the commercial monoclonal antibody, the DFA constructed in this study had more sensibility and accuracy. [Conclusions] The optimized VHH-EGFP gene could be efficiently expressed in E.coli BL21(DE3) system, and the purified recombinant fluorescent antibody was more effective comparable to the commercial monoclonal antibody, and it can be produced with a cheaper and easier process. [ABSTRACT FROM AUTHOR]

O objetivo deste estudo foi determinar a freqüência de anticorpos anti-Neospora caninum em vacas e fetos provenientes de municípios do sul de Minas Gerais. Foram utilizadas 559 amostras de soros de vacas de um banco de soros formado a partir de amostras coletadas em 18 propriedades produtoras de leite, distribuídas por sete municípios da microrregião de Lavras, MG; e 575 amostras de soros de vacas e 503 de soros fetais, ambas coletadas no matadouro de Campo Belo, MG. As amostras foram submetidas à reação de imunofluorescência indireta (RIFI-IgG), para pesquisa de anticorpos anti-N. caninum, considerando como ponto de corte o título de 1:200 para as fêmeas bovinas e 1:25 para os soros fetais. A freqüência média global de anticorpos anti-N. caninum em vacas leiteiras das fazendas da microrregião de Lavras foi de 91,2% (510/559) e nas vacas abatidas em matadouro e fetos de 97,2% (559/575) e 12,7% (64/503), respectivamente. Esses resultados indicam que a infecção por N. caninum está amplamente distribuída em rebanhos leiteiros da microrregião de Lavras, sul de Minas Gerais.The objective of this study was to determine the frequency of antibodies anti-Neospora caninum in cows and fetuses from Municipalities of southern Minas Gerais. A total of 559 serum samples of cows of the a bank of sera formed from of serum samples collected at 18 dairy farms, distributed for seven Municipalities of the Lavras Micro region, southern Minas Gerais, and 575 serum samples of cows and 503 serum samples of fetuses were collected at slaughter house of Campo Belo, MG. Serum samples were submitted to the indirect fluorescent antibody tests (IFAT), using as antigen taquizoites of N. caninum, and as cut-off values the serum dilution of 1:25 for the fetuses serum and 1:200 for the cows. The frequency in the cows of the Lavras Micro region was of 91.2% (510/559). The frequency was of 97.2% (559/575) and 12.7% (64/503) for females and fetuses from slaughter house, respectively. These results indicate that the infection by N. caninum is endemic in dairy cattle of the Lavras Micro region, southern Minas Gerais, Brazil.

Background: We have reported that handheld confocal laser endomicroscopy (CLE) can be used with various nonspecific fluorescent dyes to improve the microscopic identification of brain tumor and its boundaries. Here, we show that CLE can be used experimentally with tumorspecific fluorescent labeling to define glioma margins in vivo. Methods: Thirteen rats underwent craniectomy and in vivo imaging 21 days after implantation with green fluorescent protein (GFP)labeled U251 (n = 7) cells or epidermal growth factor receptor (EGFR) overexpressing F98 cells (n = 6). Fluorescein isothiocyanate (FITC) conjugated EGFR fluorescent antibody (FITCEGFR) was applied for contrast in F98 tumors. Confocal images of normal brain, obvious tumor, and peritumoral zones were collected using the CLE system. Benchtop confocal microscopy and hematoxylin and eosinstained sections were correlated with CLE images. Results: GFP and FITCEGFR fluorescence of glioma cells were detected by in vivo visiblewavelength fluorescence CLE. CLE of GFPlabeled tumors revealed bright individual satellite tumor cells within peritumoral tissue, a definitive tumor border, and subcellular structures. Imaging with FITCEGFR labeling provided weaker contrast in F98EGFR tumors but was able to delineate tumor cells. Imaging with both methods in various tumor regions correlated with standard confocal imaging and clinical histology. Conclusions: These data suggest that in vivo CLE of selectively tagged neoplasms could allow specific interactive identification of tumoral areas. Imaging of GFP and FITCEGFR provides realtime histologic information precisely related to the site of microscopic imaging of tumor. [ABSTRACT FROM AUTHOR]

Reasons for performing study A comprehensive evaluation of the real-time PCR assay for leptospirosis in comparison with other diagnostic assays on a large-scale basis is fundamental in validating the assay and determining the causes of equine abortions. Objectives To compare and evaluate the diagnostic value of real-time PCR assay for leptospirosis with traditional methods in equine leptospiral abortions. Study design Cross-sectional observational study. Methods A Leptospira spp. fluorescent antibody test ( FAT), microscopic agglutination test ( MAT) and real-time PCR (targeting the LipL32 gene) were compared and evaluated in equine fetal necropsy specimens (placenta, kidney, liver and heart blood) and maternal serum (when available) in 339 equine fetuses. Results From a total of 339 equine fetuses necropsied, 21 cases (6.19%) were diagnosed as leptospiral abortion. The majority of leptospiral abortions occurred in January (8 cases) and February (5 cases). Real-time PCR detected 21 of 21 cases, whereas MAT and FAT detected 19 and 18 (including 2 suspicious cases) cases, respectively. Comparing tissues, placenta yielded somewhat similar cycle of threshold values by real-time PCR compared with kidney, whereas kidney was the best specimen for the diagnosis of leptospirosis by the FAT test. In all MAT positive cases, the predominant titre in fetal heart blood was to serovar Pomona (ranging 1:100 to 1:204,800) with little or no cross-reaction to serovar Grippotyphosa. Conclusions The results indicate that real-time PCR is an effective method for the diagnosis of leptospiral abortion in horses. However, MAT should continue to be used in clinical cases for serovar determination. [ABSTRACT FROM AUTHOR]

Background: The performance of Giardia diagnostic tests that detect either cysts or fecal antigens has not been thoroughly examined. Hypothesis/Objectives: We examined the concordance and agreement among 4 Giardia diagnostic tests (2 cyst and 2 coproantigen detection methods) in a colony of dogs chronically and subclinically infected with Giardia. Animals: Twenty dogs with chronic, subclinical Giardia infection. Methods: Giardia diagnostic tests were performed repeatedly on each dog over 120 days. Fecal cyst detection methods (ZnSO4 flotation and fluorescent antibody [FAB] coproscopy) were performed 3 times per week. Coproantigen methods ( Giardia SNAP test and quantitative ELISA) were performed weekly. Results were analyzed and compared among methods. Results: When compared with FAB coproscopy, all of the in-house diagnostic tests had excellent positive predictive values (PPVs, 95–99%) at the study prevalence (89%). At lower prevalence rates, ZnSO4, SNAP, and ELISA tests all had good negative predictive values (NPVs), but poor PPVs. There was poor to good agreement among tests by κ analysis. Conclusion and Clinical Relevance: Our findings show that most commonly used in-house Giardia diagnostic tests have poor agreement with the gold standard method (FAB coproscopy). The in-house tests have good NPVs, but poor PPVs, at prevalence rates common in most clinical settings. [ABSTRACT FROM AUTHOR]

The article discusses a study on the optimization of immunohistochemical (IHC) and fluorescent antibody (FA) techniques for the detection of foot-and-mouth disease virus (FMDV) in animal tissues. The critical components of a successful IHC technique are tissue fixation and epitope (antigen) retrieval. It notes that virus-positive cells were identified by the IHC and FA techniques within the dermal papillae. According to the authors, the IHC and FA techniques used in the study could facilitate several aspects of FMDV investigation.

A direct, microscopic fluorescent antibody method was developed to detect the occurrence of Enterococcus faecium in coastal aquatic environments and was compared with the conventional membrane filtering method. The “in situ” application of the antibody-based protocol in the analysis of water samples collected from coastal polyhaline habitats demonstrated good sensitivity and ease of implementation. Data obtained with the microscopic technique were in agreement with those obtained from culture counts. The fluorescent antibody method proved to be a rapid and reliable technique for the detection of E. faecium. The advantages and limitations intrinsic to the method are discussed, highlighting the potential of this new technique for monitoring coastal aquatic environments. [Copyright &y& Elsevier]

A faecal pollution monitoring of coastal Messina waters was performed by comparing three (microscopic, enzyme, and culture) methods. Evidence of Escherichia coli cells (29.99 to 96.79% of the total enteropathogenic serotypes) retaining their viability into the marine environment was shown. β‐Glucuronidase activity rates suggested that living cells were also metabolically active. Heavily polluted sites were detected, where improperly treated urban wastes were discharged. Significant relationships between microscopic and enzymatic data proved both methods to be suitable alternatives to the culture method for E. coli detection, improving environmental quality assessment. [ABSTRACT FROM AUTHOR]

Abstract: A rapid DNA extraction and quantitative, real time polymerase chain reaction (QRTPCR) analysis method targeting the ureA gene of Helicobacter pylori was evaluated for the measurement of these organisms on membrane filters at levels that might be expected to be found in drinking water samples. No interference was seen from high levels of background organisms and related, non-target species were detected at approximately 4–5log10 lower levels of sensitivity than H. pylori by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with a species-specific fluorescent antibody and solid phase cytometry analyses. The mean detection sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of ±0.57log10 based on duplicate analyses of the samples. One liter drinking water samples from several locations in the US were inoculated with the same H. pylori cell suspensions used to generate the standard curve and gave measurements that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method. This method may be useful for the rapid screening of drinking water for H. pylori. [Copyright &y& Elsevier]

The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P. vulgaris were studied in silty loam and clayey soils. The experiments were carried out to address the performance of two rhizobia strains [CE3 and Ph. 163] and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt. Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars. The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight. This response was also positive in dry matter and biomass accumulation by the plants. The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph. 163. However, the mixed inoculant strains were not always as good as single strain inoculants. The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting. The nodule occupancy by inoculant strain Ph. 163 in both soils occupied 30–40% and 38–50 of nodules of cultivar Bronco. The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph. 163 in clayey soil. The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar. For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains. Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean. To cite this article: H. Moawad et al., C. R. Biologies 327 (2004). [Copyright &y& Elsevier]

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex. [ABSTRACT FROM AUTHOR]

Fluorescent antibody (FA) preparations were developed and tested as an approach to recognition and enumeration of a specific unicellular bluegreen alga in a natural ecosystem. Antibodies made against an axenic culture of Synechococcus cedrorum Näg. gave a high agglutination titer and an excellent homologous staining reaction. Antibody specificity was tested with 28 heterologous cultures of bluegreen algae, known cultures of chemoautotrophic bacteria and heterotrophic bacteria, and randomly selected unknown bacteria from soil, sewage and aquatic habitats. FA staining of bluegreen algae was restricted to S. cedrorum and six heterologous isolates, all indistinguishable microscopically from S. cedrorum. The ability to enumerate S. cedrorum FA-reactive cells in nature was demonstrated with data on population densities for samples taken from water columns of stratified holomictic lakes in northcentral Minnesota. [ABSTRACT FROM AUTHOR]

The article offers information on a study which explores the use and preparation of flourescent antibodies against canine distemper virus (CDV). It notes that the conventional protocol was used to develop monoclonial antibody (McAb) against CDV. It mentions that flourescein isothiocyanate labelled the McAb using dialysis methods.