Radioimmunoassay (RIA)
Radioimmunoassay (RIA) is an immunoassay that uses antigens labeled with radioisotopes in a stepwise formation of immune complexes. Radioimmunoassay is a very sensitive in vitro assay technique that requires specific safety measures, thus very rare in clinical laboratories. However, this method measures substances concentration such as those of antigens, for instance, blood hormone level through antibodies.
Although this method is rarely applied, it is the cheapest to use for such measurements. When used, licensing and special precautions are needed due to radioactive substances used, especially with the existence of ELISA that substitutes the radioisotopes with enzymes.
The assay operates in such a way that; a known quantity of an antigen is made radioactive repeatedly by marking it with isotopes of iodine that are gamma-radioactive, for example, 125-1 attached to tyrosine. This radiolabeled antigen is mixed with an unknown antibody amount for the antigen leading to the two bindings together. The free antigen is then separated by the use of a suitable technique such as precipitating it with ammonium sulfate or dialysis or using an antibody; then, centrifugation is done or filtration. The antibody radioactivity bound to it is determined in a scintillation counter, and this is considered as a control experiment. Another mixture is prepared with the same amount of antibody and similar concentration of antigen, which is radioactive plus the sample with an unknown amount of antigen. The radioactivity in this mixtures antigen is also determined. The more unmarked the antigen in the sample, the less radioactivity is bound to the antigens. Therefore, the difference in radioactivity between the sample versus the control is proportional to the quantity of the unmarked antigen in the sample. In short, the concentration of unknown antigen, which is unmarked (non-radioactive), is determined through a comparison of its inhibitory effects on the binding of antigen that is radioactively labeled with the inhibitory effect of known standards.
GnRH Antagonist Usage
Radioimmunoassay has been applied in gonadotropin-releasing hormone antagonists. GnRH is a hormone secreted from the hypothalamus by the GnRH neurons in a pulsatile pattern. It controls gonadotropin hormones, luteinizing hormone, and follicle-stimulating hormone secretion from the anterior pituitary. RIA is exploited to show the conformation of various bioactive GnRH peptides (type II′ â consisting of residue 5-8) and the pivotal role that particular residues play in binding and activation of various GnRH receptor. Yahalom et al., 2000 employ RIA to study the structural activity of reduced –sized GnRH agonist that was derived from a sequence of endothelial agonist. The study revealed a notable interaction of peptide I with the GnRH receptor that promoted the understanding of the link between GnRH and its specific receptors. Also, using the RIA, the study designed stable analogues with high potency that can be exploited to develop novel drugs capable of stimulation of GnRH secretion from the pituitary gland. The procedure begins with the peptide synthesis, followed by binding of the peptides to the GnRH receptor of the pituitary, peptide binding to the endothelin receptor, luteinizing hormone release from cultured and dispersed pituitary cells and lastly in vivo release of the luteinizing hormone. This study conducted by Yahalom et al., 2000 concluded that the GnRH receptor recognizes the cyclic conformation of peptide I. This was evidenced by the enhanced GnRH receptor affinity and increased cyclic peptide III peptides bioactivity over the bioactivity of the cyclic peptides I and II.
Similarly, Jiang et al. 2001 show that RIA can be used to determine GnRH analogues which are more soluble than the above stated and hypothesize formulations such as azaline B and acyline and increase hydrogen bonding sites on the peptides. The study shows the duration of inhibition of LH release by the GnRH analogues. While conducting a study to show a new generation of long-acting analogues which incorporate the p-Ureido-phenylalanines at Positions 5 and 6† ( GnRH antagonists), Jiang et al. 2001 used in both the reported gene assay and in vitro histamine release assay to test the selected study analogues. It showed that a series of GnRH antagonists ha urea/carbamoyl functionalities, which play a crucial role in increasing the probable intra- and intermolecular hydrogen bonding chances for structural stability and also interactions of peptides and receptors. Besides, it showed analogues that had increased hydrophilicity and reduced propensity to form a gel when subjected in an aqueous solution compared to azaline B, which resulted in increased duration of action when they were administered subcutaneously.
Usually, GnRH antagonists manage sex-steroid dependent pathophysiologies, ovulation inductions, endometriosis, and other cancers as well as male contraception. The GnRH antagonists are effective in these cases because they can inhibit gonadotropin secretion. However, they are short-acting and stimulate histamine release, and long-acting preparations are gaining interest in the management of these conditions. Unfortunately, most of the long-acting preparations such as acyline and azaline B forms gel, the most potent except for abarelix forms gel at unacceptable concentration when administered subcutaneously, and their inadequate or lack of solubility and bioavailability necessitates the nee to prepare GnRH antagonist that can last longer than a month for management of sex-hormone dependent pathophysiologies. This led Jiang et al. 2001 to use RIA to determine GnRH analogues which are more soluble than the above stated and hypothesize formulations such as azaline B and acyline. RIA enables the researchers to induce and increase the number of peptide sides’ hydrogen bonding sites to increase the structural standing and improve peptide and receptors interactions.