Localization and quantitative analysis of antigen-antibody binding on 2D substrate using imaging NanoSIMS.

Iron containing-antigen bound specifically to antibody immobilized on a surface is analyzed by nanoscale secondary ion mass spectrometry (NanoSIMS). This technique is well adapted compared with X-ray photoelectron spectroscopy and energy dispersive spectroscopy, which do not allow the detection of iron. The obtained Fe(+) map gives a good representation of the antigen repartition on the surface. NanoSIMS analysis of competition experiments performed with albumin and iron-free antigen are in good accordance with results obtained by a classical fluorescence microscopy approach. These results underline the interest of imaging NanoSIMS as a label-free method, allowing the localization and quantitative analysis of antigen-antibody binding with better spatial resolution than imaging ellipsometry and SPR.

Structures and rheological properties of hen egg yolk low density lipoprotein layers spread at the air-water interface at pH 3 and 7.

Low density lipoproteins (LDL) from egg yolk have a classical structure of lipoprotein with a core of neutral lipids surrounded by a monolayer of apoproteins and phospholipids. This structure collapses during adsorption and all constituents spread at the interface. To understand better the nature of the interactions between apoproteins and lipids at the interface, we have deposited LDL at an air-water interface and analysed the isotherms during their compression on a Langmuir trough. Then, these LDL films were studied by atomic force microscopy (AFM) imaging. To identify the protein and lipid structures, we imaged films before and after lipid solubilisation by butanol. To study the interactions in the LDL films, we have varied the pH, ionic strength and used simplified model systems. We also studied the correlation between observed structures and interfacial rheology of the film. The isotherms of interfacial LDL films were similar for pH 3 and 7, but their structures observed in AFM were different. At surface pressures below the transition corresponding to the demixion of apoprotein-neutral lipid complexes, the LDL film structure was not governed by electrostatic interactions. However, above this surface pressure transition (45mN/m), there was an effect of charge on this structure. Around the transition zone, the rheological properties of LDL films at pH 3 were different as a function of pH (viscous at pH 3 and visco-elastic at pH 7). So, the rheological properties of LDL films could be linked to the structures formed by apoproteins and observed in AFM.

Structure modification in hen egg yolk low density lipoproteins layers between 30 and 45 mN/m observed by AFM.

We have studied the structure of films made by low density lipoproteins (LDL) from hen egg yolk, which are composed of apoproteins, neutral lipids and phospholipids. These LDL have been deposited on air-water interface to form a monolayer which has been compressed to measure an isotherm using Langmuir balance. This isotherm presented three transitions (neutral lipid (surface pressure, pi=19 mN/m), apoprotein-lipid (pi=41 mN/m) and phospholipid (pi=51 mN/m) transitions). We have studied only the apoprotein-lipid transition. In order to observe the LDL film structure before (pi=30 mN/m) and after (pi=45 mN/m) the apoprotein-lipid transition, the formed films were transferred and visualised by atomic force microscopy (AFM). Our results have shown that the structures observed in the LDL film were different depending on the surface pressure. The apoproteins and neutral lipids appeared to be miscible up to the apoprotein-lipid transition, when demixing occurred. The structures observed after the apoprotein-lipid transition should be due to the demixing between apoproteins and neutral lipids. On the other hand, apoproteins and phospholipids seemed miscible whatever the surface pressure. Hence, the first transition (pi=19 mN/m) should be attributed to the free neutral lipid collapse; the second transition (pi=41 mN/m) should be attributed to the demixing of apoprotein-neutral lipid complexes; and the last transition (pi=51 mN/m) should be attributed to phospholipid collapse or to demixing of apoprotein-phospholipid complexes.

Hen egg yolk low-density lipoproteins film spreading at the air-water and oil-water interfaces.

Hen egg yolk is largely used as an ingredient in food emulsions due to its exceptional emulsifying properties. Low-density lipoproteins (LDL) are the main egg yolk constituents and the most important contributors to yolk emulsifying properties. To better understand the LDL adsorption mechanism and spreading at the interface, we extracted and studied LDL at different interfaces. At the air-water interface, the LDL film isotherm presents three transitions, and two were identified by each lipid class present in LDL. The last transition should be due to apoproteins-lipids complexes. During LDL adsorption, the presence of apoproteins at the LDL surface and the neutral lipid core is necessary. At pH 3 and pH 7, LDL are disrupted and spread quasi-similarly at the air-water interface, contrary to the oil-water interface where LDL spread more at pH 7 than at pH 3.