Publication Abstract

Development and application of an ELISA for a sex pherome released by the sea lamprey (Petromyzon marinus L.)

S.S. Yun, A.P. Scott, M. Seifkes and L. Weiming

An Enzyme-linked Immunosorbent Assay (ELISA) for a sea lamprey sex pheromone was developed and successfully applied in the study of sea lamprey reproductive biology. The conjugated bile salt, 7α, 12α, 24-trihydroxy-3one-5α-cholan-24-sulfate (commonly referred to as 3keto-petromyzonol sulfate [3KPZS]), has been identified as a sex pheromone which is released by the reproductively mature male sea lamprey and acts as an attractant for sexually mature females. A polyclonal antiserum has been raised against the pheromone by injecting 3keto-petromyzonol-24-hemisuccinate (3KPZ-HS) conjugated to Bovine Serum Albumin into rabbits. An enzyme label has been prepared by conjugation of 3KPZ-HS to acetylcholinesterase. ELISAs were performed on high binding 96-well microtiter plates. The diluted antibody and enzyme label yielded a standard curve in a working range of 20 pg/well to 10 ng/well. The intra-assay variation was less than 5% while the inter assay variation was 11.98%. The cross-reactivity of the antiserum to related compounds such as petromyzonol, petromyzonol sulfate, allocholic acid, cholic acid and taurolithocholic acid sulfate was shown to be less than 2%. The assay was successfully applied to the measurement of pheromone concentrations in water washings collected from lamprey holding water and in HPLC fractions of water washing extracts. The immunoreactive material in HPLC extracts was predominantly found in the expected elution position of 3KPZS. There was only one other peak of immunoactivity (which was relatively small and did not correspond to the elution position of unsulfated 3KPZS). It was established that this peak could be removed from the water extracts with diethyl ether – resulting in a highly specific ELISA for 3KPZS. Spermiating male sea lamprey were shown to produce and release 3KPZS at between 130 µg to 1 mg h^-1 animal^-1. This assay has applications in both physiological and ecological aspects of sea lamprey reproduction.

Reference:

S.S. Yun, A.P. Scott, M. Seifkes and L. Weiming, 2002. Development and application of an ELISA for a sex pherome released by the sea lamprey (Petromyzon marinus L.). General and Comparative Endocrinology, 129: 163-170.