Innobiochips - Understanding life

Vianney SOUPLET, Christophe OLIVIER, InnoBioChips, Institute of Biology of Lille, Oleg MELNYK, Rémi DESMET, UMR 8161 CNRS, University of Lille Lille 1 and 2, Pasteur Institute of Lille, IFR 142

Microarrays are miniaturized analytical devices. On a flat solid substrate of a few cm², such as microscope glass slides or the bottom of a 96-well plate, probe biomolecules are arrayed using a robot. These probes can be peptides, (1) proteins, (2) antibodies or polysaccharides. (3) They are used to capture target biomolecules present in the sample. Target capture is detected by various methods, with Fluorescence being the most widely used. Peptide and protein microarrays offer many applications such as the detection of infections, study of autoimmune diseases, screening of sample banks, simultaneous profiling of proteins,  disease markers or the monitoring of immunization as it is the case here.

In Alzheimer's disease, brain tissue presents senile plaques which are due to an abnormal accumulation of a peptide, the amyloid Aβ1-42 peptide. A strategy to combat this disease is to develop a vaccine capable of inducing a production of antibodies specific for Aβ1-42 peptide or certain derivatives, and thus leading to a reduction or even the elimination of senile plaques. It is essential in this type of research to characterize, precisely, the specificity of the antibodies produced after immunization.

To achieve this, it is important to compare the affinity of the antibodies produced for a large number of peptide probes. However, as immunization experiments are conducted on mice, the serum volume  that can be collected by bleeding is low, i.e. only a few microliters. This is where the analytical power of peptide microarrays in a single analysis proves to be of great interest.

We have, in this way, developed a peptide microarray with a series of decapeptides descended from the amyloid Aβ1-42 peptide. The sequence of various peptides is shifted forward by one amino acid on the sequence of peptide Aβ1-42.  The decapeptides are called 1 to 11 according to the position of their first amino acid sequence of peptide Aβ1-42. On the microarray, each line contains four replicates with the same concentration. A column is made up of different concentrations of the same peptide of 10-6 M (upper spots) to 10-4 M (lower spots). To form a plot, the robot lays down 1 nanoliter, from a sample of 1 microliter, in a source plate.

Image of biochips incubated in the presence of mouse serum, and secondary antibody labeled with a fluorophore. These 16-bit images are quantified using appropriate software. An analysis in triplicate can provide the results as medians and interquartile ranges (Work done in collaboration with Nicolas SERGEANT and Marie GOMPEL in the Neurodegenerative Diseases and Neuronal Death team managed by Luc Buée of Unit U837 of the Jean-Pierre Aubert Center).

Serum was collected from mice before (Fig. 1A) and after immunization (Fig. 1B). An analysis requires 0.5 microliter of serum. The microarray is incubated with the diluted serum, washed and then incubated with a labeled antibody by a fluorophore specifically recognizing the mouse antibodies captured on the plots. The biochip was read using a fluorescence scanner, which provides a resolution of a few micrometers. The color of the image reflects the intensity of fluorescence. Before immunization, only the control plots IgG and prot A are visible. After immunization, peptides 1-4 provide a strong signal; they contain the epitope that was used for immunization. The signal for peptide 5 is lower, which is normal, as the epitope is reduced by 1AA. The peptide 6(-2AA) does not give a signal that is stronger than the background noise of the biochip.  A weak signal reappears for peptides 7 and 8 (cross-reactivity of antibodies induced by immunization).

On a microarray, the size of plots (≈ 100 µm) can hold a large number of different probes on a surface of only a few mm². Thus, peptide and protein microarrays are expected to revolutionize the field of biological analysis, along the lines of the impact of DNA microarrays on genomics research. The miniaturization of these systems enables them to conduct a complete study in only a few days, while a classical experimental approach would take several weeks or even months of work.

Miniaturization also provides a gain in detection sensitivity compared to classical macro tests such as ELISA (Enzyme Linked Immunosorbent Assay), used routinely for the serodetection of infections. (4, 5) A study conducted at the Institute of Biology of Lille showed the performance of peptide and protein microarrays for the serodetection of multiple infections by HIV, HCV, HBV and Treponema pallidum, the bacterium responsible for syphilis. (2, 6)

Here, the experiment requires a very small amount of probes and biological samples. Thus, miniaturization proves to be a major asset when analyzing samples that are rare or available in small quantities. The serodetection experiment presented above is a perfect illustration of this point.

Reading systems are becoming more sophisticated and hence adding an extra dimension to analysis by microarray. In fluorescence, detection is done by using scanners capable of detecting emission at several wavelengths simultaneously. The combination of the presentation of a large number of probes and reading at several wavelengths opens up new prospects for biological analysis

The recent technological developments concern the use of devices in 96-well plastic with a biochip at the bottom of each well, and the development of chemical methods of binding peptides or proteins to the plastic substrate. (7) The combination of the  microarray format and 96-well format, detection sensitivity, use of small amounts of a biological sample, reading at several wavelengths and, finally,  the use of incubation and standard wash systems for 96-well plates make microarray technology for peptides and proteins a major asset for biology research.

Bibliography

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(2) Duburcq, X., Olivier, C., Malingue, F., Desmet, R., Bouzidi, A., Zhou, F., Auriault, C., Gras-Masse, H., and Melnyk, O. (2004) Peptide-protein microarrays for the simultaneous detection of pathogen infections. Bioconjug Chem 15, 307-16.

(3) Carion, O., Lefebvre, J., Dubreucq, G., Dahri-Correia, L., Correia, J., and Melnyk, O. (2006) Polysaccharide microarrays for polysaccharide-platelet-derived-growth-factor interaction studies. Chembiochem 7, 817-26.

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(5) Ekins, R. P., and Chu, F. W. (1991) Multianalyte microspot immunoassay--microanalytical "compact disk" of the future. Clin Chem 37, 1955-67.

(6) Duburcq, X., Olivier, C., Desmet, R., Halasa, M., Carion, O., Grandidier, B., Heim, T., Stievenard, D., Auriault, C., and Melnyk, O. (2004) Polypeptide semicarbazide glass slide microarrays: characterization and comparison with amine slides in serodetection studies. Bioconjug Chem 15, 317-25.

(7) Carion, O., Souplet, V., Olivier, C., Maillet, C., Medard, N., El-Mahdi, O., Durand, J. O., and Melnyk, O. (2007) Chemical Micropatterning of Polycarbonate for Site-Specific Peptide Immobilization and Biomolecular Interactions. Chembiochem 8, 315-322.