Beta-Amyloid [1-40] And [1-42] Colorimetric ELISA Kits, Human From Invitrogen

My dissertation on Alzheimer's Disease depended on measurements of beta-amyloid levels from transgenic mouse brain homogenates. To measure beta-amyloid levels, I used the Biosource International (Invitrogen) Human Beta-Amyloid [1-40] and [1-42] ELISA Kits which are immunoassays for the quantitative determination of beta-amyloid 40 and 42, respectively, in biological samples. The kits recognize both natural and recombinant human beta-Amyloid. The ELISA kits comes packaged with a pre-coated plate (with an antibody directed towards the N-terminus), beta-amyloid standard, detector antibody (directed towards the C-terminus), streptavidin-HRP, diluents, wash buffer, chromogen, stop solution and complete protocol.

After using approximately 8 kits and assaying a total of 1536 wells, I have come to trust the data generated from these kits and will rely on these kits for future projects that need assessment of beta-amyloid (Abeta) levels. The trust I have in these kits derives from the standard beta-amyloid (1-40 or 1-42) curve that the user generates from supplied synthetic Abeta. Just like the advertised standard curves in the user manual, the standard curves I generated from these kits were reproducible and robust. Although the standard curves were linear and the interwell variability within a plate was tolerable, the variability between plates is a bit high. Therefore, experimental data should be interpreted from the reading of a single plate.

In the previous version of the kit (from Biosource), the total incubation time was 6 hrs. In the newer version of the kit (from Invitrogen), the incubation time has been reduced to 4hrs. The incubation times have been reduced because the volumes required for primary antibody and secondary antibody were reduced. A brief outline of the protocol for those unfamiliar with ELISAs, prepare and dilute sample to fit within the , bind sample to the antibody coated wells, wash sample away from wells, bind secondary that recognizes a different region of the peptide, wash away excess secondary conjugated with a reporter system, wash excess secondary, and measure secondary activity. Although there are multiple steps in an ELISA, as long as one has quality reagents and treats each well equally, one can obtain reliable results. The kit comes with quality reagents at least as far as I can tell, and the little instruction booklet explains in an follow step-by-step manner.

One downside to the kit is its dynamic range: the current version of the kit has a range of 3.1-200 pg/mL, which is not as large as the older version of the kit. In the pre-Invitrogen version of the kit, the dynamic range was 15-2000 pg/mL, which allowed the user to assay a sample without as many dilutions. The second downside, obviously pertinent to all ELISA based systems, is the uniform washing that's required between changes from primary to secondary antibody solutions. If the user will perform many ELISAs, it would be recommended to invest in a modern plate washer or a multpipettor. In order to obtain uniform washing within a reasonable amount of time, an eight-tip or a twelve-tip pipet was used to dispense wash buffer into individual wells. After the wells were washed, the plate was flicked upside down in order to empty the wells for the next wash. ELISAs washing could be improved if there was an automated way of washing. I tried using an outdated BioRad automated washer but the tendency of salt crystals to form around the metal tips.

Although determining the levels of Abeta requires roughly 4 hours of time, it is 4 hours well-spent because the results obtained are reproducible and specific. Usually, 2 wells for a single measurement were used in order minimize variability. The secondary HRP provided by the manufacturer though adequate, produced a bluish color. If a yellow color is needed for detection, the ABC kit (from Vector Laboratories, cat #AK-5001) which uses strepavidin conjugated to alkaline phosphatase could be used. Alkaline phosphatase hydrolyzes a substrate termed PNPP which produces a yellow color. Hydrolysis of PNPP may be preferable because the yellowish color tends to be more permanent than the bluish color. In summay, this slight modification of the kit may be useful to some and even without this modification, these Abeta ELISA kits are an excellent choice for obtaining Abeta levels.