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Results for “antibody-based approaches”

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At least 19 recordsLinked to original sources
PubMed2026

A critical appraisal of base-resolution m6A profiling techniques.

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNA, influencing RNA fate and gene regulation. Early antibody-based approaches enabled transcriptome-wide profiling but lacked resolution and quantitative accuracy. Newer approaches now achieve base-resolution m6A detection using improved crosslinking, chemical or enzymatic conversion, and single-molecule sequencing. Antibody-free methods provide quantitative stoichiometry from minimal input, while nanopore direct RNA sequencing offers real-time, single-molecule readouts across entire transcriptomes. Collectively, these methods form a versatile toolkit that integrates global mapping with precise site-level analysis, advancing knowledge of context-dependent m6A regulation in physiology and disease. This review compares their principles, strengths, and limitations to guide method selection and highlight how next-generation epitranscriptomic tools are paving the way for clinical and therapeutic applications.

Humans↗
PubMed1985

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450.

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.

Amino Acid Sequence↗
PubMed2026

Biofilm-derived curli and Z-DNA shape anti-DNA antibody responses during Salmonella infections.

Antibodies to Z-DNA, a non-canonical DNA conformation with a left-handed zigzag backbone, are abundant in the serum of patients with systemic lupus erythematosus (SLE), with levels increasing with disease activity and flares. As SLE is associated with bacterial infections, and as extracellular DNA (eDNA) within biofilms of several bacterial species has been shown to adopt the Z-DNA conformation, bacterial Z-DNA may represent a source of immunogenic Z-DNA in SLE and other related autoimmune conditions. In these studies, we investigated whether eDNA in Salmonella biofilms also contained Z-DNA and whether such Z-DNA could elicit an antibody response. Using antibody-based staining approaches, we observed abundant eDNA in Salmonella enterica serovar Typhimurium (STm) biofilms in both the Z- and canonical B-DNA configurations, consistent with the highly Z-prone nature of the GC-rich Salmonella genome. To assess the functional contribution of these DNA conformations to biofilm integrity, biofilms were treated with DNase I, which lacks enzymatic activity against Z-DNA, or with benzonase, a nonspecific nuclease that degrades both B- and Z-DNA. DNase I treatment applied after biofilm maturation was less effective at thinning biofilms than treatment during early biofilm formation, a pattern also observed with benzonase treatment. Purified curli:DNA complexes contained Z-DNA and, when administered intraperitoneally to mice, elicited robust anti-Z-DNA antibody responses. Similarly, infection with invasive STm induced the production of anti-Z-DNA antibodies in vivo. Moreover, STm infection in mice fed a diet that promotes biofilm development was associated with increased Z-DNA levels in the cecal lumen and elevated anti-DNA antibody responses. Collectively, these findings suggest that Z-DNA, likely formed by extruded Salmonella genomic DNA, and embedded within curli:DNA complexes of STm biofilms, triggers a host immune response and drives anti-Z-DNA antibody production. This work provides mechanistic insight into how bacterial infections and diet-dependent modulation of biofilm formation may contribute to anti-Z-DNA antibody responses in autoimmune diseases like SLE.

Animals↗
PubMed1992

A simple approach to improving sensitivity in a one-step monoclonal antibody-based ELISA for human IIbIIIa using multiple conjugates.

This paper describes an approach which can be used to increase both the speed and the sensitivity of a monoclonal antibody (MAb) based ELISA for human IIbIIIa by using a mixture of enzyme labeled MAbs (conjugates) and a one step incubation format (where analyte and conjugates were incubated simultaneously for 2 h in the MAb coated well). A simple competitive blocking method is described for mapping the relative epitopes of five monoclonal antibodies (MAbs) with minimum preparation of enzyme conjugate. This method permits the selection of three MAbs which recognize distinctly different epitopes, and are suitable for use in a one step ELISA format. Compared to the regular two step ELISA using two MAbs, this simple modification improves the assay sensitivity by three-fold and decreases the incubation time by 75%, while maintaining a high level of precision (2-15%). The assay is very specific and can accurately measure both recombinant and native IIbIIIa.

Antibodies, Monoclonal↗
PubMed1991

Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies.

The quaternary structure of rat liver cytochrome P-450 within microsomal membranes from 3-methyl-cholanthrene-treated rats was examined by a novel chemical cross-linking-monoclonal antibody approach. Complex formation among the different forms of P-450 was probed by cross-linking of membrane proteins followed by immunopurification with a monoclonal antibody (mAb) to P-450c, the major 3-methylcholanthrene-inducible form. Subsequent immunoblot analysis of the immunopurified proteins with this mAb indicated that P-450c formed complexes with other microsomal proteins. Immunoblots with mAbs to different P-450s were carried out to identify the P-450s that were cross-linked to P-450c. This approach detected specific cross-linking of P-450c to P-450 2a. Immunoinhibition experiments suggest that P-450 2a further metabolizes the primary phenols produced by P-450c-catalyzed hydroxylation of benzo[a]pyrene. Complex formation among membrane-bound enzymes has implications for their catalytic efficiency and an approach combining cross-linking and monoclonal antibody-based characterization of cross-linked proteins will be useful for elucidating such membrane protein macrostructures.

Animals↗
PubMed1990

Determination of a unique and immunodominant epitope of human T cell lymphotropic virus type I.

The identification and isolation of unique and immunogenic recombinant epitopes for human T cell lymphotrophic virus (HTLV) type I might allow the development of an antibody-based assay to differentiate between HTLV-I and HTLV-II infections. To test the feasibility of this approach, an HTLV-I envelope epitope was isolated by immunoscreening of a lambda gt11 recombinant HTLV-I DNA library with a human monoclonal antibody to HTLV-I. This recombinant epitope. MTA-4, when tested with sera from HTLV-I- or HTLV-II-infected individuals, was reactive with all HTLV-I and nonreactive with all HTLV-II antisera. These results indicate that MTA-4 is a unique and immunodominant epitope on HTLV-I and confirm the usefulness of human-derived monoclonal antibodies in an experimental approach to dissect the human humoral response to a viral pathogen.

Antibodies, Monoclonal↗
PubMed1988

Newborn screening for cystic fibrosis using an enzyme linked immunoabsorbent assay (ELISA) technique.

Neonatal screening for cystic fibrosis (CF) has been conducted using conventional radioimmunoassay. An alternative immunoassay approach has been developed and applied to screening 35,550 newborns. Seventeen confirmed CF infants were detected by both assays. This monoclonal antibody-based enzyme immunoassay using microtitre plate ELISA technology has proved effective in case finding and offers a number of advantages. A reduced labour component, ease of handling, use of nonradioactive reagents, long reagent shelf-life, greater specificity and a reduction in potential sample handling and transposition errors combine to make this technology appropriate for a large volume neonatal screening laboratory.

Antibodies, Monoclonal↗
PubMed2026

Antibody-Based Proximity Labeling Reveals Bait-Proximal Proteomes in Paraffin-Embedded and Snap-Frozen Tissue Samples.

Proximity-based labeling approaches have proven highly valuable for uncovering protein-protein interactions, yet their application to primary patient material remains challenging. Here, we present a workflow enabling the use of the antibody-based ProtA-Turbo proximity labeling system in both formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue specimens. Our method efficiently directs biotinylation to diverse antibody baits across tissues of different origins. Downstream mass spectrometry-based proteomics analyses demonstrate the specificity of the method by profiling the proximal proteome of H3K27ac-marked chromatin, the nuclear lamina-associated protein EMD, and the Ser2-phosphorylated POLR2A subunit of RNA polymerase II. Using this method, we identified cell-type-specific factors and transcriptional regulators in salivary gland carcinoma, healthy testis, and testicular cancer tissue sections. The ability to detect disease-associated complexes directly within their native, spatially resolved cellular context using ProtA-Turbo can provide new insights into the molecular basis of human disease and may reveal novel, potentially actionable factors with translational relevance.

Humans↗
PubMed2026

Construction of Reverse Genetics System for Feline Calicivirus FCV-BJ616 and Proteomic Analysis.

Feline calicivirus (FCV) is a primary cause of upper respiratory tract infections and oral ulcerative disease in cats and exhibits substantial genetic diversity that complicates prevention and control. In this study, we isolated the FCV-BJ616 strain, established a reverse-genetics system, and investigated its pathogenic mechanisms, thereby providing a foundation for antibody-based therapies and broad-spectrum vaccine development. The virus was purified by three rounds of plaque cloning, and its morphology was examined by electron microscopy. VP1 expression was confirmed by immunofluorescence and Western blotting. Using integrated systems-biology and reverse-genetics approaches, an infectious clone of rFCV-BJ616 was successfully assembled and rescued, exhibiting genetic stability comparable to that of the parental strain. In vivo infection experiments showed that rFCV-BJ616 retained wild-type virulence, causing persistent high fever, weight loss, and multiorgan pathology in infected cats. Proteomic analysis indicated that infection with FCV-BJ616 or rFCV-BJ616 markedly activated cytokine-mediated inflammatory signaling pathways. Both FCV-BJ616 and rFCV-BJ616 significantly upregulated the expression of IL-8, S100A8/A9, and TLR3, which are associated with acute inflammation and tissue damage. Furthermore, elevated IFN-β levels concomitant with STAT1 downregulation suggested a transient attenuation of antiviral signaling during early immune activation. These findings were corroborated by ELISA-based validation of serum cytokine profiles. Collectively, this study provides new insights into the molecular pathogenesis and evolution of FCV-BJ616 and establishes a robust reverse-genetics platform for precise genome manipulation and future vaccine development.

Animals↗
PubMed1988

Persistence of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariasis.

This study was designed to reexamine the efficacy of diethylcarbamazine for bancroftian filariasis with special reference to changes in serum parasite antigen levels and antifilarial antibody titers after treatment. Patients with asymptomatic microfilaremia were treated with 6 mg/kg diethylcarbamazine daily for 12 days. Microfilaria counts fell dramatically after treatment, as expected. IgG antibody titers to adult and microfilarial antigens of B. malayi were increased 1 month after treatment in most patients. Titers fell slowly to or below pretreatment levels, but remained positive during subsequent months. Parasite antigen levels, measured by monoclonal antibody-based enzyme immunoassay, decreased to 72%, 58%, 53%, and 48% of pretreatment values 1, 3, 6, and 12 months after diethylcarbamazine treatment, respectively. Parasite antigen levels decreased similarly in subjects with and without residual microfilaremia after treatment. These results suggest that diethylcarbamazine has only partial macrofilaricidal activity against W. bancrofti with this dosage schedule. The sustained, impressive reductions in microfilaria counts after treatment despite significant persistence of parasite antigenemia may be explained by sublethal effects of the drug on adult worms. We believe that parasite antigen detection represents a valuable new approach for monitoring the efficacy of antifilarial drug therapy which we hope will lead to improved use of existing drugs and aid in the evaluation of new drugs for filariasis.

Animals↗
PubMed1988

Noninvasive renal diagnostic studies.

Traditional methods of noninvasively evaluating patients for renal injury do not accomplish the following tasks: reliably distinguish potentially treatable forms of acute renal failure from acute tubular necrosis; provide a sensitive indicator of early allograft rejection in renal transplant recipients, particularly those in the pediatric age group; provide an early warning of incipient drug-induced nephrotoxicity; or serve as an adequate screening test for renal injury due to exposure to occupational or environmental toxins, especially heavy metals. Because of this, considerable effort has been devoted to the development of assays to satisfy these needs. Three approaches include measurement in the urine of low-molecular-weight plasma proteins such as beta 2-microglobulin; a variety of kidney-derived enzymes, such as L-alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase; and specific renal antigens using immunologic detection. The first two of these have not proved to be adequately sensitive or specific, complicated by the frequent loss of activity associated with the physicochemical characteristics of the urine or the presence of pyuria. Despite this, useful information has been obtained. In particular, assays of beta 2-microglobulin urinary excretion and retinol binding protein appear to have clinical utility that should be pursued. Recent experience with a monoclonal antibody-based assay for a unique proximal tubular antigen, the adenosine deaminase binding protein, suggests that a battery of such assays, each directed against an antigen localized to a particular segment of the nephron, may be particularly useful.

Acetylglucosaminidase↗
PubMed1986

Use of parasite antigen detection to monitor the success of drug therapy in Dirofilaria immitis-infected dogs.

Recently the authors developed a monoclonal antibody-based enzyme immunoassay for circulating Dirofilaria immitis antigen and demonstrated its utility as a diagnostic tool for canine dirofilariasis. In the present study, serum parasite antigen measurements were used to monitor the success of thiacetarsamide therapy in 2 controlled trials that involved 24 naturally infected dogs. Parasite antigen levels correlated significantly with adult worm burdens in untreated control dogs. Antigen levels fell dramatically by 8 wk after treatment in successfully treated dogs and were undetectable 12 wk after treatment in dogs that were parasitologically cured. Microfilarial counts exhibited seasonal periodicity in both treated and control dogs and were not useful in monitoring the success of adulticide therapy. Parasite antigen detection is quite useful in monitoring the efficacy of adulticide therapy for dogs infected with D. immitis. This approach may lead to improved clinical use of thiacetarsamide, and it should facilitate evaluation of new drugs for this important infection.

Animals↗
PubMed1992

Aflatoxins isolated by immunoaffinity chromatography from foods consumed in The Gambia, West Africa.

An aflatoxin-specific, monoclonal antibody-based immunoaffinity chromatography method has been developed for the rapid isolation of aflatoxins from human foods. Aflatoxins were isolated by immunoaffinity chromatography from a variety of cooked foods, including maize, rice, millets, groundnut sauces, and leaf sauces, collected in The Gambia, West Africa. The aflatoxins were measured by direct fluorescence or high-pressure liquid chromatography. The highest levels were found for groundnut sauces, mean 162 ppb (range 18 to 943 ppb) for 18 positive samples, but aflatoxins were found in other foods; e.g., maize, mean 9.7 ppb (range 2 to 35 ppb) for nine positive samples. The food analysis results were used with records of the amounts of cooked food to estimate a mean daily intake for an individual of the order of 3.5 micrograms of aflatoxins per day. This approach for exposure assessment is considered in relation to other biomarkers of aflatoxin exposure using biological fluids.

Aflatoxins↗
PubMed1987

How sensitive are the commercial assays for anti-HTLV III/LAV?

The sensitivity of nine commercial assays, Western blot, and a newly developed monoclonal antibody-based assay for antibody to human T lymphotropic virus type III/lymphadenopathy associated virus (anti-HTLV III/LAV) were evaluated using a panel of mainly weak-positive sera. In tests on 20 sera three commercial assays and a monoclonal-based assay, representing two different solid-phase methodologies, were found to be more sensitive than Western blot. Our findings suggest that Western blot cannot be depended upon as the sole confirmatory test for anti-HTLV III/LAV. The continued use of the more sensitive enzyme-linked immunosorbent assays (ELISAs), particularly those of dissimilar methodology, would be a more valid approach to confirmatory testing.

Acquired Immunodeficiency Syndrome↗
PubMed2026

Network methods for diagonal integration of unpaired single-cell multiomics data: a review.

MOTIVATION: Advances in single-cell sequencing have enabled multiomics profiling at unprecedented resolution; however, mass spectrometry-based single-cell proteomics (scMS) remains inherently destructive, precluding simultaneous transcriptomic capture. Unlike antibody-based methods such as CITE-seq, which permit paired profiling but are restricted to targeted protein panels, scMS provides unbiased, genome-scale coverage of the intracellular proteome yet necessitates post hoc integration of unpaired datasets. This diagonal integration challenge, where transcriptomes and proteomes are measured in separate cells lacking shared anchors, remains underserved by existing reviews, which focus predominantly on vertical integration strategies enabled by non-destructive assays. RESULTS: We survey the complete computational pipeline for constructing mechanistic proteogenomic networks from unpaired single-cell data, covering: (i) unimodal network inference such as knowledge-based approaches, probabilistic graphical models, temporal directionality inference, and generative and foundation model strategies that establish the transcriptomic scaffold; (ii) cross-modal integration architectures such as network propagation, graph neural networks (scMRDR, scmFormer, scCotag), and consensus frameworks designed explicitly for the unpaired proteomics setting; and (iii) benchmarking paradigms spanning network reconstruction (BEELINE, GRETA, CausalBench) and multi-task integration evaluation (scMultiBench, SCMMIB), with guidance on metric selection under network sparsity and class imbalance. We identify three principal axes of future development: generative proteomic translation from transcriptomic precursors, inductive prior embedding in next-generation architectures, and perturbation-based causal benchmarking. AVAILABILITY AND IMPLEMENTATION: This is a review article; no novel software is distributed. A curated benchmark resource table, methods starter guide, and per-method bottleneck annotations are provided in the Supplementary Material.

Multiomics↗
PubMed1993

Glycated LDL concentrations in non-diabetic and diabetic subjects measured with monoclonal antibodies reactive with glycated apolipoprotein B epitopes.

The potential importance of the non-enzymatic glycation of low density lipoproteins (LDL) in atherogenesis and in the accelerated atherosclerosis associated with diabetes is well recognized. However, it has been difficult to evaluate LDL glycation in the clinical setting because of the lack of suitable methods. To approach this problem, we produced monoclonal antibodies, designated ES12, that recognize glycated apolipoprotein B epitopes in the LDL complex in human plasma. Here we report the use of these antibodies in a competitive enzyme-linked immunosorbent assay (ELISA) to measure glycated LDL concentrations in plasma from non-diabetic and diabetic subjects. In this assay, glycated LDL in the soluble phase inhibits binding of the ES12 antibody to glycated LDL immobilized to microtitre wells, whereas other glycated proteins and non-glycated LDL do not compete. A linear dose-response relationship for 10-125 ng glycated LDL per well allows the construction of standard curves, from which the concentration of glycated LDL in human plasma can be determined. The mean concentration of glycated LDL in samples from non-diabetic subjects was 21.8 +/- 0.9 mg/l, increasing to 40.8 +/- 2.6 mg/l in samples from patients with type II diabetes, comprising 1.9-4.8% and 3.2-14.8%, respectively, of total apolipoprotein B. Glycated LDL concentrations in samples from diabetic patients correlated positively and significantly with other indices of glycaemic status. The results indicate that circulating glycated LDL, which may have diagnostic and pathophysiologic importance, is increased in diabetes with attendant hyperglycaemia. The results further indicate that the described monoclonal antibody-based competitive ELISA affords a simple and reproducible method for quantitative measurement of glycated LDL.

Animals↗
PubMed1984

Application of acrylamide as an embedding medium in studies of lectin and antibody binding in the vertebrate retina.

The use of acrylamide as an embedding medium for vertebrate retinal tissue and its applicability to lectin and antibody-based cytochemical studies is described. The acrylamide technique has numerous advantages over those using fresh-frozen or paraffin embedded material. The morphological integrity of retinal tissue prepared in acrylamide is equivalent to that obtainable with paraffin and superior to that of fresh-frozen material. In addition, this technique alleviates problems often encountered with the thermal and chemical treatments required in the paraffin method. The acrylamide technique allows the localization of lectin and antibody-binding sites essentially unaltered by the fixation and embedding protocol, as in frozen sections, while maintaining tissue morphology similar to that of paraffin-embedded tissue. It is hoped that this approach will be useful to other workers in vision research employing lectin, antibody or other cytochemical approaches to the study of cellular structure and function.

Acrylamide↗