Questions:

What kind of data will the S&S Serum Biomarker Chip provide?

Four. Each of the two dual-pad glass slides consists of two identical antibody microarrays.

Two dual-pad experiments per Serum Biomarker Chip kit.

The Serum Biomarker Chip is intended to be used with human serum samples. However, the chip can most likely be used with almost any biological material from which you can isolate proteins, such as plasma, tissues, and cells.

All of the antibodies on the S&S chips are specific to human proteins. However, some of the printed antibodies may also recognize the corresponding mouse and rat homologous proteins. The Serum Biomarker Chip is not designed for comparing protein expression between different species.

The green and red fluorescent signals from a spot on the chips correspond to the amount of labeled protein captured from the two samples applied to the chip. These values are used to calculate a ratio between the two serum samples. The ratio will tell you if the protein is more or less abundant in one sample versus the other sample. The profile obtained with the 120 antibody specificities results in a differential pattern that discriminates between the two serum samples.

The advantage of the microarray format over conventional protein detection assays is the ability to quickly and easily determine how the abundance of 120 proteins has changed between two samples to identify which groups of proteins or patterns can be identified. The two serum samples may represent either disease vs. healthy or two similar but different disease states. The chip is not designed to quantify the exact amount of protein bound and has not been validated to quantify protein measurements. The Serum Biomarker Chip provides some indication as to which serum biomarkers are over- or under-abundant between the two serum samples. Therefore, as with any qualitative screening tool, we recommend validating the changes in the biomarker abundance with an additional technique such as S&S FAST Quant® or an ELISA.

One experiment requires eight microliters of test serum and eight microliters of control serum.

Each spot of the microarray contains approximately 0.75 - 1.0 nanogram of either a polyclonal or monoclonal antibody.

The protocol uses common laboratory equipment and techniques.

The Serum Biomarker Chip is designed for use with the reusable Chip Clip™ Slide Holder (Item Number 10486081) and the S&S Two Color Labeling and Fluorescent Detection Kit (Item Number 10486085). The Two Color Labeling and Fluorescent Detection Kit contains sufficient materials to process one Serum Biomarker Chip kit (two dual-pad slides).

A fluorescent scanner compatible with 75 x 25 x 1 mm FAST Slides and capable of measuring DY™647 and DY™547 fluorescent labels is required for excitation and analysis. Dy547 has an absorbance maximum of 557 nm and an emission max of 574 nm. Dy647 has an absorbance maximum of 652 nm and an emission max of 673 nm. S&S recommends the Axon GenePix® 4100A Personal Scanner to scan the processed slide. We recommend the use of ArrayVision™FAST® software to spot-find, sample the data, and export the ratio-metric data as a Microsoft Excel file.

Yes. The fluorescent dyes and spin columns are included in the S&S Two Color Labeling and Fluorescent Detection Kit.

Many of the antibodies have been validated using FAST Quant® immunoassay technology. All antibodies have been tested for reactivity with the appropriate immunizing antigen. We test reproducibility of our antibody chips by calculating the average Dy™547/Dy™647 ratio over eight experiments. The CV averages 16.1%.

We have detected approximately 1 ng/ml of known proteins spiked into whole serum prior to labeling. Detection of different proteins in serum will depend on the affinity of the antibody for the antigen, the prevalence of the antigen in the mixture and the number of labeling sites on the antigen.

No.

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Chip Clip™ Slide Holder, S&S® and FAST® are trademarks of Schleicher & Schuell BioScience, Inc.

ULS™ is licensed from and a registered trademark of KREATECH Biotechnology B.V., and covered by one or more patents of KREATECH Biotechnology BV, including, but not restricted to, the following: EP 0,539,466; US 5,580,990; US 5,714,327; US 5,985,566; US 6,133.038; and EP 1,019,420.

DY™647 and DY™547 are registered trademarks of Dyomics GmbH. DY™647 is covered by the license of Dyomics GmbH, Germany, to produce and distribute dyes for in vitro applications claimed by the patent family DE 44 45 065.

GenePix™ is a registered trademark of Axon Instruments© 2004 Schleicher & Schuell BioScience

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