Introduction

The most basic event, in 96-microwell plates applications, is the interfacial interaction (adsorption) between the solid surface and proteinaceous molecules in a fluid phase. The details of the interaction and, ultimately, the performance and quality of the plate, are largely dictated by its surface properties.

Polystyrene (PS), because of the hydrophobic nature of its surface and the excellent optical properties, has become the material of choice for the production of microtiter plates. Later on, the eminently hydrophobic interfacial interaction has been strengthened by the introduction of small amounts of polar groups in the PS surface,a finding which resulted in the coming of age of so called enhanced binding or high binding capacity microtiter plates.

In this study, we use the sub-nm resolution of the Atomic Force Microscope (AFM) to obtain informations on the interfacial interaction between conventional or enhanced binding properties of microtiter plates surfaces and proteins. AFM can image molecules adsorbed to a surface. The obtained images are affected by a number of variables (including the strength of the substrate-molecule interaction) and, in principle, could allow to appreciate the different interfacial behavior of immunomolecules on conventional Vs. enhanced binding surfaces.

Experimental

The adsorption behavior of rabbit anti-human IgM and its binding with human IgM was evaluated on standard and enhanced binding Biomat microtiter plates (details described below, in the relevant section).

AFM analysis was performed using a Nanoscope III AFM, Digital Instruments, USA. The AFM was operated in the contact mode. Samples were imaged using a Si₃N₄ cantilever, with a spring constant of 0.12 N/m and a 125x125 µm scanner.

IgM capture experiments on standard and HB Biomat microtiter plates.

Standard (untreated) and HB (surface treated) PS microtiter plates were coated overnight at 4 °C with 100µl of affinity-purified rabbit anti-human IgM (A426, DAKO, Denmark) diluted 1:2000 in 0.1 M carbonate-bicarbonate buffer, pH 9.6.

After three washes with 0.1 M phosphate buffered saline (PBS), pH 7.2, 100 µl of human IgM (BIO-SPA Milan, Italy) diluted in 0.1M PBS + 0.05% Tween^® 20 were added to the above coated plate. Uncoated wells were used as controls.

The solutions were incubated for 15 minutes at room temperature. After three washes with PBS and three washes with distilled water, the plates were dried for 60 min. at 37 °C, foil packaged under vacuum and stored at 4 °C until use.

Coated surfaces were imaged by AFM.

fig. 1 surface of control HB (neither Anti-IgM, nor IGM) fig. 2 surface of coated standard (Anti-IgM + IGM) fig. 3-4-5 surface of coated HB (Anti-IgM + IGM) fig. 6 surface of HB exposed to the IgM solution without Anti-IgM coating. fig. 7 IgM drawing	fig. 1	fig. 2	fig. 3
	fig. 4	fig. 5	fig. 6
	fig. 7

Results

AFM results show a completely different behaviour of standard and HB plates towards Anti-IgM, IgM adsorption. In the former case (fig. 2 ), AFM reveals that something lies on the surface, but no meaningful images are obtained. This is a typical AFM artefact, which occurs when the cantilever tip of the AFM probe scrapes and removes a loosely bound surface layer. The mechanism is sketched below (the arrow indicates the direction of the AFM probe-sample motion):

fig.8

On the other hand, the AFM image of the Anti-IgM, IgM coated HB surface (fig. 3) shows a markedly different morphology. A closely-packed array of similarly shaped units apparently paves the well surface. Their shape is more clearly seen in fig. 4-5 . Note that the size of the field of view is some tens of nm, while the vertical scale is only a few nm. It is possible to appreciate that the units paving the surface have an approximately pentagonal shape, with a region of very low density in the middle, i.e. exactly the expected shape of IgM molecules (fig. 7). The AFM detected size of the molecules is about 38 nm, in good agreement with the reported value of 30 nm (because of the finite dimension of the AFM tip, it is known that the width and length of nm-size objects is a little overestimated by the AFM).

Beside the resolution at the molecular level shown in these photographs, it is important to appreciate the different response of standard and HB to AFM analysis. In the former case (fig. 2), the Anti-IgM, IgM layer is too loosely bound and cannot be imaged. HB surfaces (fig. 3) allow a stronger bond, and molecular resolution images of adsorbed IgM are detected (note that in the control HB -fig.1- , exposed to the IgM, Tween-containing, solution but not subjected to the Anti-IgM coating, no adsorbed layer is observed).

Conclusions

In conclusion, the AFM analysis allowed to image IgM molecules on Anti-IgM, IgM coated, HB (surface treated) microplates. On the other hand, the standard (untreated) PS plate was not able to establish a strong bond with the molecules of the immunocomplex, and the loosely bound surface layer could not be imaged by AFM.

Beside offering molecular-level resolution of adsorbed molecules, these results confirm the important role of surface modification in microtiter plates performances.