EIAs and ELISAs are convenient, high-sensitivity, peroxidase-based technologies that enable the amount of protein present in a sample to be measured in a single day. Novel lysis reagents have been developed (Lysis Reagents 1 and 2) to facilitate simple and rapid extraction of cellular molecules.
The Biotrak™ cAMP competitive enzyme immunoassay system is specifically designed for research purposes. The kit includes protocols using novel lysis reagents in order to facilitate simple and rapid extraction of cAMP from cell cultures. These components avoid the requirement for traditional, time-consuming extraction procedures and eliminates the need for removal of extraction reagents prior to measurement. It combines the use of a peroxidase-labeled cAMP conjugate, a specific antiserum which can be immobilized on to precoated microplates, and a one-pot stabilized substrate solution.
Lysis reagent 1 hydrolyzes cell membranes to release intra-cellular
cAMP. Lysis Reagent 2 sequesters the key component in Lysis
Reagent 1 and ensures cAMP is free for subsequent analysis. The
detergent/sequestrant complex does not interfere with antigen:
antibody binding. Lysis Reagent 1 is simply added to cultured cells,
followed by a 5 to 10 min incubation before assay. The
antiserum is reconstituted with Lysis Reagent 2. The assay is based
on competition between unlabeled cAMP and a fixed quantity of
peroxidase-labeled cAMP, for a limited number of binding sites on a
cAMP specific antibody.