Lateral Flow Serodiagnosis in the Double-Antigen Sandwich Format: Theoretical Consideration and Confirmation of Advantages

Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Serodiagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production, thus increasing the amount of attention in a tense epidemiological situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen-specific antibody-labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally.

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.