Beta Amyloid, 1-42 And 1-40 ELISA Kits From Signet Laboratories (A Covance Company)

Signet Laboratories’ ELISA kits for the measurement of beta amyloid 1-40 or beta amyloid 1-42 are quantitative sandwich ELISAs. These ELISAs are designed to measure beta amyloid 1-40 or beta amyloid 1-42 in cell culture supernatants, serum and plasma. The assays take 4.5 hours to perform after an initial overnight incubation with standards and samples. The ELISAs use an antibody specific for either beta amyloid 1-40 or beta amyloid 1-42, which has been pre-coated onto a 96-well individual strip microplate. A more detailed description of the procedure follows, but in general: Standards (recombinant human beta amyloid 1-40 or beta amyloid 1-42) and samples are added to the wells and any beta amyloid 1-40 or beta amyloid 1-42 present is bound by the immobilized antibody. After washing the microplate to remove any unbound substances, a primary antibody specific for the carboxy terminus of either beta amyloid 1-40 or beta amyloid 1-42 is added to the microplate wells. After incubation, the microplate is washed once again, this time to remove unbound antibody. At this point, a secondary antibody conjugated to HRP is added; this secondary antibody binds to the primary antibody. Another wash is performed after which the substrate solution is added to the wells and color development is allowed to proceed. Color development occurs in proportion to the amount of beta amyloid 1-40 or beta amyloid 1-42 bound in the initial step. After an incubation period, the color development is stopped and the intensity of the color is measured.

We have used this assay primarily to measure levels of beta amyloids 1-40 and 1-42 secreted from cell lines into cell culture media. All reagents are brought to room temperature before beginning the assay. The first step in either ELISA is to prepare the standards: A beta amyloid 1-40 or beta amyloid 1-42 lyophilized peptide standard is reconstituted in 1 ml of standard diluent (supplied) to make a 10000 ng/ml solution. This is then further diluted with wash/sample diluent (supplied) to give a concentration of 100 ng/ml. Once the beta amyloid 1-40 or beta amyloid 1-42 standard is prepared, dilutions are prepared in wash/sample diluent to generate beta amyloid 1-40 or beta amyloid 1-42 standards from 1000 pg/ml to 15.63pg/ml in doubling dilutions. The ELISA plate is removed from its foil pouch and washed twice with the wash/sample diluent. At this point, 100 ul of each standard (in duplicate) and each sample are added to the ELISA plate. The ELISA plate is then incubated overnight at 4-8ºC. After the overnight incubation, the plate is aspirated and washed three times with the wash/sample diluent. Then 100 ul of anti-beta amyloid 1-40 or anti-beta amyloid 1-42 is added to each well of the plate and incubated for 2 hr at room temperature. After the incubation, the plate is aspirated and washed 3 times. The next step is to add 100 ul secondary antibody-HRP conjugate to the plate which is then incubated for another 2 hrs.

The plate is again washed 3 times prior to the addition of 200 ul of the OPD substrate solution. The substrate solution is prepared by adding two 10mg OPD tablets to the supplied OPD diluent, followed by the addition of 50 ul of 3% H₂O₂. The plate is then incubated for 20-25 minutes in the dark while the color develops. The color development is stopped with the addition of 100 ul of the stop solution (3 N sulfuric acid); the plate can then be read in a microplate reader at 492 nm.

A standard curve is created from the standards on the plate and this is used to calculate the concentration of beta amyloid 1-40 or beta amyloid 1-42 in the samples. There are many types of software which will perform this calculation; we have used Molecular Devices SOFTmax® Pro. It is important to keep the amount of beta amyloid 1-40 or beta amyloid 1-42 within the dynamic range of the assay. Samples may need to be diluted to bring them within that range if, on first measurement, they have an elevated beta amyloid 1-40 or beta amyloid 1-42 level. We have found the intra- and interassay precision to be very robust. In conclusion, Signet Laboratories’ ELISAs for beta amyloid 1-40 and beta amyloid 1-42 are simple, fast and reliable kits.