Biomarkers of oogenesis in Macoma balthica determined by subtractive immunization

We employed the technique of subtractive immunization (SI) to identify specific biomarkers of oocyte development in Macoma balthica. Analysis revealed that 2 novel proteins (45 and 96 kDa) were induced during female gametogenesis that were neither present during other life stages, nor in male clam tissues. A total of 78 viable hybridomas were specific for either the 45 or 96 kD M balthica proteins. Subsequent peptide fragment analysis of the 96 kDa protein revealed that it was essentially identical (>99.9%) to the evolutionarily conserved heat shock protein 70 family. The resultant hybridomas were screened for their utility in a quantitative capture ELISA configuration. Two antibody systems were designed that allowed the quantification of either the 45 or 96 kDa protein in M. balthica tissue homogenates and are useful for studies on M balthica gametogenesis and fecundity. Subtractive immunization is a powerful tool, as it simultaneously identifies novel protein targets while producing monoclonal antibodies specific to them.