Purification of vitellin from grass shrimp Palaemonetes pugio, generation of monoclonal antibodies, and validation for the detection of lipovitellin in Crustacea

Abstract

Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68–85 kD and two high molecular mass bands in the range of 190–221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.