For several years, Alzheimer’s Disease research has experienced a hotbed of activity in the pharmaceutical industry. A specific target of interest has been beta-amyloid (Abeta) peptides, which are suspected to aggregate into plaques that progressively inhibit neuronal activity resulting in clinical dementia as well as death in some cases. What makes Abeta such a hard target is the peptide hydrophobicity and the multitude of Abeta peptides produced by the enzymes in question (although two chief Abeta peptides, Abeta1-40 and Abeta1-42, are usually targeted for analysis, others can be found endogenously). As can be expected, the pathway for beta amyloid production is being rigorously pursued as a viable therapeutic target by many research institutions. The research toward understanding beta-amyloid is so aggressive and popular that Ciphergen has put together a kit to use in conjunction with it’s SELDI-TOF line of mass spectrometers.
The rigorous method is outlined step-by-step, however, it only works with ProteinChips that are compatible with the SELDI-TOF mass analyzer system. The process is based on surface-immunoprecipitation, with the provided antibody coupled to the surface of the provided ProteinChips (Protein A/G surface). Twelve (PS20) ProteinChip Arrays are included, each having eight sample spots. The provided antibodies (6E10 and IgG for negative control) are diluted per the instructions and applied, allowing time to incubate. The antibody is then deactivated with a provided solution and washed with two other provided solutions (PBS with detergent followed by PBS alone). The sample is then applied and incubated on the spot (the spots can hold 5 uL although as much as 20 uL has been added in this laboratory) without rocking. Bioprocessors are available from Ciphergen that allow up to 400 uL of fluid to be incubated (with rocking), which usually goes overnight at 4C. After incubation, the sample is removed and the ProteinChip spots are carefully washed with the same solvents as used above (PBS/detergent, PBS), followed by a quick wash with HEPES (concentrated HEPES included for further dilution). As in MALDI-TOF sample preparation, an energy absorbing matrix is applied and allowed to dry in order to crystallize the target for SELDI analysis (alpha-cyano-4-hydroxy-cinnamic acid is provided). Ciphergen also includes positive controls (individual tubes containing 10 uL of 1uM lyophilized Abeta1-40, Abeta1-42, and Abeta1-16) for system validation and instructions for creating a simple standard curve (N=1). These standards are also useful for method optimization and calibration of the SELDI instrument.
The results obtained in this laboratory have typically not been as good for real samples as they are for the Abeta standards in buffer. The wash steps seem excessive, which may cause concern for precious biofluids. The mouse monoclonal antibody provided comes from an ambiguous source bearing a Ciphergen label, but 6E10 from other sources has been used with similar results. Aside from these shortcomings, the kit is relatively inexpensive for the material provided and if such a kit may be useful in your own research, we would recommend trying the kit for use with SELDI-TOF technology. Some biofluids may provide better results than others, but we have not screened all possible sources of Abeta to date so another laboratory’s results may vary from our experience.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research