Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 7678631

Conformation-sensitive immunoassay: optimisation, validation and evaluation.

S Lolov·S Tyutyulkova·I Marinova·I Kehayov·S Kyurchiev
View original source ↗Export metadata ↓

Abstract

A conformation-sensitive immunoassay (CSI) has recently been developed (Pfund and Bourdage, 1990). This paper describes the optimal laboratory protocol which makes this method useful as an early screening procedure for conformation-sensitive monoclonal antibodies. The method was validated with a panel of 12 monoclonal antibodies. Results from our experiments confirm that this variant of CSI can be applied in the analysis of purified proteins and glycoproteins, as well as complex mixtures of antigens.

Explore related subjects

Antibodies, MonoclonalEpitopesHumansImmunoassayProtein ConformationJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

S Lolov, S Tyutyulkova, I Marinova, I Kehayov, S Kyurchiev. 1993-01-04. Conformation-sensitive immunoassay: optimisation, validation and evaluation.. https://doi.org/10.1016/0022-1759(93)90065-f

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

ZrO₂@C-based colorimetric/photothermal dual-mode immunosensor coupled with a novel monoclonal antibody for quantification of Aspergillus ochraceus biomass.

Aspergillus ochraceus contaminates agricultural products and produces nephrotoxic, carcinogenic ochratoxin A (OTA), posing severe food safety hazards. A dual-signal lateral flow immunochromatographic assay (dLFIA) based on ZrO₂@C nanoprobes was established for quantitative detection of A. ochraceus biomass. A novel monoclonal antibody (mAb 4B4) was prepared as the capture antibody to immobilize A. ochraceus mycelial lysate antigen on the test line, and a rabbit polyclonal antibody (pAb G2801) as the detection antibody to modify ZrO₂@C composites (synthesized via UiO-66 pyrolysis) into 200 nm colorimetric/photothermal nanoprobes. This dLFIA achieved limits of detection of 0.164 μg/mL (colorimetric) and 0.517 μg/mL (photothermal). This efficient and reliable method allows quantitative analysis of A. ochraceus biomass, which is suitable for routine monitoring of fungal contamination in agro-food matrices.

Antibodies, Monoclonal↗
PubMed1998

Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase.

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.

Antibodies, Monoclonal↗
PubMed1998

Identification of two domains of the p70 Ku protein mediating dimerization with p80 and DNA binding.

The Ku autoantigen is a heterodimer of 70 (p70) and approximately 80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1-115 and 430-482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430-609), but not interaction with p80. The p70 mutants 1-600, 1-542, 1-115, and 430-600 did not bind DNA efficiently. However, DNA binding of 1-600, 1-542, and 1-115, but not 430-600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the N-terminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.

Antibodies, Monoclonal↗