Summer Research
* Student(s): Matthew Van Hook , Robert Wysocki
Advisor: Herman Lehman
Title: Development of a cAMP Enzyme-Linked Immunosorbent Assay (ELISA)
Abstract:
In invertebrate systems, the neurotransmitter octopamine (OA) stimulates the production of adenosine- 3, 5 cyclic monophosphate (cAMP) through a G-protein coupled receptor. Our research developed a competitive enzyme-linked immunosorbent assay (ELISA) to measure levels of cAMP produced by a Sf9 cell line from the ovaries of the fall armyworm, Spodoptera frugiperda. The development of this assay will allow for the analysis of dose-response characteristics of the OA receptor when stimulated by OA compared with the dose-response of the receptor when stimulated with an aldehyde or ketone derivative of OA. The number of assays needed to effectively characterize the OA receptor makes the use of a commercially available cAMP ELISA kit excessively expensive, thus prompting our desire to produce a comparable assay for a lower price.
The development of the ELISA involved refining the operational concentrations of the various components needed. We first coupled cAMP to horseradish peroxidase (HRP) by means of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide which created a bond between the amine groups of the HRP and a carboxyl group on a modified cAMP 2 -O-succinyl cAMP. We obtained a binding curve with this conjugate, and determined that it should be used at a concentration of 1/10,000 in the assay. Additionally, we obtained a binding curve with a range of primary antiserum dilutions, determining an optimal dilution of 1/6,000. We next developed an acetylated standard binding curve with cAMP concentrations spanning from 10-7 M to 10-12 M, giving us an IC50 of 0.0015 pmol/50ul compared to an IC50 of 0.05 pmol/50ul (also acetylated) from a cAMP enzyme immunoassay kit (Assay Designs, Inc.) and an IC50 of 1.27 pmol/25ul (non-acetylated) from Lombardi, et al (2004). Our ELISA was not sensitive to non-acetylated cAMP. An ATP cross-reactivity test of our ELISA showed that it was approximately 1% sensitive towards ATP, compared with 0.33% from the Assay Designs, Inc. ELISA kit. In summary we produced an ELISA that was comparably cross-reactive to ATP, and more sensitive to cAMP than the commercially available kit.
1. Lombardi VC, DA Schooley. A method for selective conjugation of an analyte to enzymes without unwanted enzyme-enzyme cross-linking. Analytical Biochemistry. 331 (2004) 40-45.
Research by MVH supported by the Sergei S. Zlinkoff Fund for Medical Education. Research by RW supported by the Ralph E. Hansmann Science Student Support Fund.