Louise D. Gerber

Scintillation proximity assay: a sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions

Scintillation proximity assay (SPA) makes it possible to use radioisotopes for monitoring binding reactions continuously without the need to separate free from bound components. As a result SPA can be carried out more rapidly than most other methods used to monitor binding reactions. The methodology also lends itself to automation. The sensitivities already achieved with SPA procedures are comparable to the sensitivities of other procedures in use today. Another feature of SPA is that the key reagents (beads, 125I labeling) are relatively inexpensive. The principles of SPA, utilizing 125I-labeled molecules, are discussed and some applications to immunology, receptor binding, and measurement of potential across membranes are presented. SPA should also be applicable to monitoring interactions involving nucleic acids, lipids, and carbohydrates. Characteristics of some radionuclides, other than tritium and 125I, that may be used in SPA are presented.

Isolation and characterization of the opioid peptides from rat pituitary: beta-lipotropin

Rat beta-lipotropin was isolated from 40 anterior pituitaries using high efficiency chromatography columns and sensitive fluorometric methods. The beta-lipotropin was characterized as to molecular weight, amino acid composition, and generation of opioid activity by trypsinization. Other opioid peptides, as well as a precursor to opioid peptides with a molecular weight larger than that of beta-lipotropin, were observed in this tissue.

Binding assay for opioid peptides with neuroblastoma × glioma hybrid cells: Specificity of the receptor site

A sensitive and rapid radioreceptor assay has been developed to monitor opioid peptides in column effluents. It is based on competitive binding to NG 108-15 cells using [3H-Tyr]Leu-enkephalin as the displaced ligand. The specificity of binding to the cells of naturally occurring opioid peptides and synthetic analogs has been shown to be similar to that found with synaptic plasma membranes.

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.

Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: Application to placental alkaline phosphatase

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.