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GFP-SCFV: expression and possible applications as a tool for experimental investigations of atherosclerosis.

Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA s

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus.

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysa

The blockade of IL-6 signaling in rational drug design.

After three decades from the development of the hybridoma technology, a monoclonal antibody-based therapy targeting the inflammatory cytokine has been established as an ultimate treatment for chronic inflammatory diseases. Interleukine-6 (IL-6) is one of the inflammatory cytokines playing a pivotal role in these conditions, and strategies targeting IL-6 signal show promise in the treatment of chronic inflammatory diseases such as rheumatoid arthritis, juvenile idiopathic arthritis, and Crohn's disease. Although many groups have been exploring the approach to block the IL-6 signal, tocilizumab,

Differential chromatin looping regulates CD4 expression in immature thymocytes.

Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, using looping chromatin immunoprecipitation and chromatin conformation capture assays, we demonstrated that interactions between Runx1 and positive elongation factor b (P-TEFb) appose the silencer and enhancer in CD4-negative thymoma cells and double-negative immature thymocytes. This chromatin loop decoys P-TEFb away from the promoter, thus preventing RNA polymerase II from elongating on the CD4 gene. In the absence of Runx1 on the silencer, P-TEFb interacts with the transcription complex, forming

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine.

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease o

Production of monoclonal antibodies against partially purified surface tegument antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) were produced by the in vitro fusion of Balb/C mice spleen cells immunized with partially purified surface tegument antigens of Fasciola gigantica. The surface membrane and tegument antigens were purified by using gel filtration chromatography. SDS-PAGE performed on the processed proteins demonstrated that the proteins had molecular weights of 20 to 97 kDa. In this study, fifteen monoclonal antibody clones were selected from the hybridoma clones, namely: 1B7, 1B11, 1B12, 1C9, 1D4, 1G2, 1H7, 2B6, 2C3, 2C9, 2D11, 2F11, 2G2, 2G5, and 2G11. They were evaluated by immun

Single, antigen-specific B cells used to generate Fab fragments using CD40-mediated amplification or direct PCR cloning.

A simple procedure for the generation of human antibody fragments directly from single B cells or B-cell clones is described here. The procedure is based on antigen-specific selection of single human B cells, using antigen-coated magnetic beads and a cellular amplification step based on a culture system involving both EL-4 thymoma cells and anti-CD40 antibodies, presented by CD32-expressing fibroblasts. Nested PCR was applied to rescue V-regions from both single B cells and B-cell clones obtained using the cellular amplification step. This amplification step both increased the cell number as w

Serodiagnosis of human paragonimiasis by ELISA-inhibition test using monoclonal antibodies.

ELISA-inhibition test using Paragonimus westermani specific monoclonal antibody (Mab) was investigated to improve the diagnostic specificity of paragonimiasis. By cell fusion, one hybridoma clone secreting anti-P. westermani specific Mab was selected (Pwa-14), which reacted on bands of 28 kDa, 42.5 kDa, 89 kDa and 120.5 kDa. IFA showed Pwa-14 was located at the vitelline follicles. By micro-ELISA, 100% of 22 paragonimiasis cases were found positive, but 5 of 40 clonorchiasis cases (12.5%), 3 of 26 cysticercosis cases (7.7%) showed false positive. None of 10 sparganosis patients or 28 normal co

Interleukin-6 in serum of patients with polymyalgia rheumatica and giant cell arteritis.

Polymyalgia rheumatica and giant cell arteritis are diseases which are characterized by a brisk acute phase response. Cytokines, principally interleukin-1 and interleukin-6, are thought to mediate the release of acute phase proteins from hepatocytes. Employing a sensitive bioassay based on the hybridoma growth factor properties of interleukin-6, we investigated its levels in sequential sera obtained from 15 initially untreated patients with PMR/GCA. We found that interleukin-6 activity was significantly elevated in all untreated patients. Although there was a significant decline in its levels

Enzyme-linked immunosorbent assay of serum pepsinogen I.

A hybridoma monoclonal antibody against human pepsinogen I was used to develop an enzyme-linked immunosorbent assay for pepsinogen I in serum. In the two-step competitive procedure using antimouse immunoglobulin F(ab')2 fragment coupled to alkaline phosphatase, the measurable assay range was 8-256 micrograms/l. No cross-reactivity with rat pepsinogen 1, human pepsinogen II, gastrin I, bombesin, somatostatin and peptide YY was shown. However, there was slight cross-reactivity (0.09%) with porcine pepsinogen. The coefficients of variation within and between series were 7.6% and 13.0%. This enzym

A mouse monoclonal IgE antibody anti bovine milk beta-lactoglobulin allows studies of allergy in the gastrointestinal tract.

A monoclonal IgE antibody directed against bovine milk beta-lactoglobulin (beta-LG) was produced by fusion of NSI myeloma cells with spleen cells of Balb/c mice immunized with alum-precipitated beta-LG. This antibody was found by radioimmunoassay to react with both native and aggregated beta-LG, but a positive passive cutaneous anaphylaxis reaction (PCA) was evident only with aggregated beta-LG. 1 ng of this purified antibody was capable of eliciting a PCA. The chemical and physical properties were characterized by amino acid and carbohydrate analysis and by ultracentrifugation. The epsilon-ch

The diagnostic distinction between malignant mesothelioma of the pleura and adenocarcinoma of the lung as defined by a monoclonal antibody (B72.3).

The correct distinction between malignant mesothelioma of the pleura and adenocarcinoma of the lung has become increasingly complex, with a variety of histochemical, immunohistochemical, and ultrastructural studies to be performed on biopsy material. The reliability of immunohistochemical studies has been hampered by the use of polyclonal antisera to "carcinoembryonic antigen (CEA)" and keratin. Hybridoma technology now offers monoclonal antibodies (MAbs) in unlimited quantity and standardized quality to selective ranges of specific antigenic determinants. MAb B72.3, generated against a membra

Monoclonal antikeratin antibody: production, characterization, and immunohistochemical application.

A monoclonal antikeratin antibody, EKH4, was produced from a hybridoma cell line which was established by fusing P3X63SAg8 mouse myeloma cells with spleen cells of mice immunized with human trichilemmoma cells. Immunoblot analysis showed that EKH4 antibody reacts predominantly with 50 kilodalton keratin polypeptide in normal epidermis. By indirect immunofluorescence and immunoperoxidase techniques, EKH4 antibody reacted with the lower 2-3 cell layers of the epidermis as well as most cells of pilosebaceous follicle of human and animal skin. Tumor cells of human basal cell epitheliomas and squam

LILRB2 inhibition by blocking antibody ES009 reprograms immunosuppressive myeloid cells and enhances T-cell-mediated tumor immunity.

The inhibitory receptor leukocyte immunoglobulin-like receptor B2 (LILRB2), expressed on myeloid cells like monocytes and tumor-resident myeloid cells, sustains an immunosuppressive tumor microenvironment and promotes tumor progression. Phase 1 clinical studies of LILRB2-targeting drug candidates have shown favorable safety, dose-dependent target engagement, and preliminary antitumor activity, both as monotherapy and in combination with anti-PD-1. These findings support the blockade of LILRB2 to reprogram myeloid cells and enhance immunotherapy. Building on this, we have developed ES009, a nan

Structure and function of therapeutic antibodies approved by the US FDA in 2025.

In 2025, the 50th anniversary of the invention of hybridoma technology, the United States Food and Drug Administration (US FDA) approved a total of 44 new molecular entities (NMEs), of which 10 were therapeutic antibody-based molecules. Additionally, the US FDA approved 31 small molecules (of which one was a peptide), and 3 new nonantibody therapeutic proteins, but no new chimeric antigen receptor (CAR)-T-cell therapeutics. Of the new antibody-based biologics, two were antibody-drug conjugates, one was a T-cell engager-bispecific antibody, two were Fc-modified, half-life extended antibodies, a

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… Introduction–antibodies approved by US FDA in 2025 This year, 2025, marks the 50th anniversary of the first paper, written by Georges Köhler and César Milstein, describing the methodology for generating a monoclonal antibody (mAb) by using their newly created hybridoma technology [ 1 ]. … The variable regions of six of the newly approved antibodies were derived from human genes, and four were humanized from mouse hybridomas and then humanized ( Table 1 ). …

Development of a monoclonal antibody-based time-resolved fluorescence immunochromatographic assay strip for rapid and quantitative determination of S100B in serum.

Traumatic brain injury (TBI) represents a significant global health challenge requiring rapid and accurate diagnostic tools. S100 calcium-binding protein B (S100B) serves as a consensus-endorsed serum biomarker for TBI, but current detection methods lack the speed and simplicity needed for on-site clinical applications. This study reports the development of a time-resolved fluorescence immunochromatographic test strip (TRFIS) for rapid and quantitative serum S100B detection. High-affinity monoclonal antibodies against S100B were generated via hybridoma technology, and time-resolved fluorescent

Screening and identification of B-cell epitopes on the p72 protein of African swine fever virus.

African swine fever (ASF) is a highly contagious and virulent infectious disease caused by infection of pigs with the double-stranded DNA virus African swine fever virus (ASFV). The p72 protein is the major component of the ASFV icosahedral capsid, comprising approximately 32% of the viral particle, and is essential for viral adsorption and cellular entry. In this research, we expressed the p72 protein using a eukaryotic system, and it exhibited strong reactivity with ASFV-positive porcine serum. We developed eight monoclonal antibodies using hybridoma technology, six of which recognized the n

Highly Specific Monoclonal Antibodies Combined with A Multiplex Quantum Dot Microsphere-based Fluorescence Lateral Flow Immunochromatographic Assay for Simultaneous Detection of Vanillin, Methyl Vanillin, and Ethyl Vanillin in Food Samples.

Vanillin (VAN), methyl vanillin (VER), and ethyl vanillin (EVA) are widely used as flavor enhancers in processed foods. Monitoring their levels is essential to prevent overuse or adulteration, especially in infant foods. Herein, a computational chemistry-aided molecular modeling technique was employed to design specific haptens for VAN, VER, and EVA. High-affinity monoclonal antibodies were generated via hybridoma technology with matrix effect-enhanced screening, yielding half-maximal inhibitory concentrations of 22.10, 49.94, and 40.84 ng/mL. A multiplex quantum dot microsphere-based fluoresc

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, m

Development of an anti-Pfs230 monoclonal antibody as a Plasmodium falciparum gametocyte blocker.

Vector control is a crucial strategy for malaria elimination by preventing infection and reducing disease transmission. Most gains have been achieved through insecticide-treated nets (ITNs) and indoor residual spraying (IRS), but the emergence of insecticide resistance among Anopheles mosquitoes calls for new tools to be applied. Here, we present the development of a highly effective murine monoclonal antibody, targeting the N-terminal region of the Plasmodium falciparum gametocyte antigen Pfs230, that can decrease the infection prevalence by > 50% when fed to Anopheles mosquitoes with gametoc