Assays for oxytocin detection

What is Oxytocin?

Oxytocin is a nine amino acid cyclic peptide with 2 disulfide loops made in the hypothalamus and controlled by the posterior pituitary. It has been termed the Love hormone because the concentration increases with social bonding, kissing, cuddling, and sexual intercourse. It also stimulates uterine contractions during pregnancy in a positive feedback loop, and it plays a significant role in maternal bonding and milk production in mammals. In males, oxytocin seems to be involved in competitive responses and the ability to fight or flee during confrontations. This could be because oxytocin behaves differently in the male and female amygdala.

There has been considerable increased interest in the effects of oxytocin, especially in the social behavioral field. PubMed citations have increased almost 2-fold over the past 10 years. Oxytocin is derived from the 10 kDa protein neurophysin 1 encoded by the human OXT gene. It is highly conserved in mammals.

In fish the equivalent hormone is isotocin in which 2 amino acids are switched. The glutamine is replaced by serine in isotocin, and leucine is replaced by isoleucine.

In nonmammalian tetrapods such as birds, reptiles, and amphibians, mesotocin substitutes for oxytocin. Mesotocin has an isoleucine replacement for the leucine in oxytocin. In fish, isotocin takes the place of oxytocin.

Detecting Oxytocin with assay kits

Arbor Assays collaborated with Prof. Evan MacLean and his student, Gitanjali Gnanadesikan at the University of Arizona Canine Cognition Center, to determine the binding site of our antibody. In the published study, urine samples from wildtype and knockout mice were tested using our Arbor Assays DetectX Oxytocin EIA kit, K048-H1/H5.

The authors, including one from Arbor Assays, investigated various oxytocin fragments, including OT 2-9, OT 4-9, OT 7-9, and melanocyte-inhibiting factor1 (MIF-1) as potential cross reactants in the assay. They also used epitope mapping to investigate where the antibody binds. Pepscan Presto BV in the Netherlands was contracted to construct arrays of synthetic peptides to scan all the amino acid sequences for oxytocin and neurophysin I prepropeptide, the precursor for oxytocin. Here are the results from one of the Pepscan studies using the Arbor Assays kit rabbit antibody:

There is some non-specific binding of the detection system used, but there is definitive identification of the major binding regions. The study rationalized the antibody binding in relation to the cross-reactivity data:

The antibody in the assay kit recognizes the sequence from amino acids 2-9. The increase in binding for the assay to oxytocin versus OT 2-9 suggests that the disulfide bridge between 1 and 6 generates a 3-dimensional antibody site which enhances the binding.