Immunological relatedness of annelid extracellular hemoglobins and chlorocruorins

1. The immunological relatedness of several annelid extracellular hemoglobins and chlorocruorins was investigated using ELISAs and Western blotting to determine the binding of purine polyclonal and monoclonal antibodies to Lumbricus terrestris hemoglobin with the hemoglobins of Tubifex tubifex, Tylorrhynchus heterochaetus, Arenicola marina and Macrobdella decora and the chlorocruoins of Myxicola infundibulum and Eudistylia vancouverii. 2. Polyclonal antibodies to Lumbricus terrestris hemoglobin bound to all the other hemoglobins and chlorocruorins. However, the titers were in all cases one to several orders of magnitude smaller than with Lumbricus terrestris hemoglobin. 3. Polyclonal antibodies to Eudistylia vancouverii chlorocruorin bound to the hemoglobins of Lumbricus terrestris, Tubifex tubifex, Arenicola marina, Tylorrhynchus heterochaetus and Macrobdella decora. 4. Of the nine monoclonal antibodies to Lumbricus terrestris hemoglobin isolated, two (No. 24 and No. 26) bound to the other hemoglobins and to Myxicola chlorocruorin, but the binding was again weaker than with Lumbricus hemoglobin. Antibody No. 26 also bound to Eudistylia chlorocruorin. Although antibody No. 24 appears to recognize a conformation-dependent epitope, antibody No. 26 recognizes a common epitope in each of the four subunits M, D1, D2, and T of unreduced Lumbricus hemoglobin. 4. An additional two monoclonal antibodies to Lumbricus hemoglobin (No. 21 and No. 25) bound also only to Tubifex hemoglobin. Antibody No. 21 recognizes subunits D1 and M of Lumbricus hemoglobin and No. 25 appears to recognize a conformation-dependent epitope.