Two novel enzyme-linked immunosorbent assays and NMR studies of the immunodominant repeat unit of the circumsporozoite protein of the malaria parasite Plasmodium falciparum

A novel two-site enzyme-linked immunosorbent assay (ELISA) which utilizes a murine monoclonal antibody (MAb) antigen (Ag) capture system and does not require purified enzyme-linked MAb in the detector system has been developed. This assay may be applied to the detection of malarial sporozoites using an MAb directed against the circumsporozoite (CS) protein of Plasmodium falciparum (MAbpf). Antigen captured by MAbpf bound to the wells of a polystyrene microtiter plate may be detected spectrophotometrically using a stable pre-formed complex between purified or crude species-specific MAbpf and affinity purified goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (GAMIgG.HRP). Bound antigen is quantitated based on a standard curve of purified protein generated per assay. A mass ratio of 1:2 for MAbpf: GAMIgG.HRP was found to maximize the signal-to-noise ratio. The limit of detection for this assay is 100 sporozoites. Another two-site antigen capture ELISA has been developed. This spectophotometric assay provides a quantitative measure of the murine monoclonal antibody concentration of a specific subclass contained in cell culture supernates, based on a standard curve of purified antibody generated per assay. The assay utilizes heterogeneous goat anti-mouse immunoglobulin G (GAMIgG) as the coating antibody and GAMIgG.HRP as the detector antibody. A sensitivity as low as 2 ng of heterogeneous mouse immunoglobulin (hMIgG) was achieved with this assay. One dimensional NMR spectroscopy was utilized in an examination of the solution-state secondary structure of the repeating unit, Asn-Pro-Asn-Ala, of the immunodominant region of the P. falciparum circumsporozoite protein. This study was initiated in order to experimentally test the theoretical prediction of a type I $\beta$-turn structure for the tetrapeptide, due mainly to the presence of $\beta$-turn inducing residues, asparagine and proline, at positions one, two, and three. The N-acetylated tetrapeptide amide, N-Acetyl-Asn-Pro-Asn-Ala-NH$\sb2$, was synthesized by the solid phase technique and subsequently analyzed for secondary structure conformation in two aqueous solvent mixtures, water/deuterium oxide and water/deuterated trifluoroethanol. NMR data indicate that the predominant structure formed in solution for the N-acetylated tetrapeptide amide is a half-turn.