Schultz et al.: Immunologic methods for species identification of early-life-stage lutjanid fishes 



741 



KDa 



205- 



116- 

 97.4- 



66- 



45- 

 29- 



Figure 6 



SDS-PAGE of the dissolved precipitates from the am- 

 monium sulfate fractionation (0.38 mg/mL) of soluble 

 extracts of 1) L.jocu; 2) L. apodus; and 3) L. griseus. 

 Total protein added to each lane was 235 ug. The open 

 triangle ( ) shows the position of the 66 kDa pro- 

 tein; the closed triangle ( 4 ) marks bands not seen in 

 L. griseus (lane 3). The gel was stained with Coo- 

 massie brilliant blue. 



polyclonal and monoclonal antisera for identifying 

 specimens at an early life stage. Animals will be im- 

 munized with highly purified proteins to enhance 

 possibilities of producing antibodies to minor struc- 

 tural protein differences. The use of highly purified 

 protein is especially important for monoclonal anti- 

 body production because immunization with complex 

 mixtures of antigens generally results in monoclonal 

 antibodies being produced to immunodominant 

 epitopes (Barclay and Smith, 1986; Matthew and 

 Sandrock, 1987 ). Immunization with a highly purified 

 protein increases possibilities of producing monoclonal 

 antibodies with a fine degree of specificity for each spe- 

 cies of lutjanid. It remains for future experiments to 

 determine if these specific antisera will also be species 

 specific for life history specimens, such as eggs, larvae, 

 and early juveniles. Although the anti-66 kDa IgG used 

 in these studies also reacted with the early life history 

 specimens of L. griseus (Fig. 2), future experiments with 

 monoclonal antibodies generated to the 66 kDa pro- 

 tein and other lutjanid proteins (Fig. 6) will determine 

 their usefulness as specific reagents. 



In other studies, the use of a very pure antigen 

 was successful for predator-prey studies with 

 Sciaenops ocellatus (red drum) (Schultz and Clarke, 

 1995; Arnold et al., in press), in which a specific 

 polyclonal antiserum was prepared by immunizing 

 a goat with a highly purified 80 kDa glycoprotein. 



Monoclonal antibodies were used successfully to 

 detect epitopes in a number of marine and fresh 

 water species. For example, An et al. (1990) used a 

 low molecular weight eluate from SDS-PAGE slab 

 gels of a protein extract from rock shrimp to produce 

 highly specific murine monoclonal antibodies. A 

 monoclonal antibody to partially purified mature egg 

 proteins reacted with eggs, embryos and larvae of 

 Asterina pectinifera but not with other species be- 

 longing to the same genus or in mixtures of biologi- 

 cal specimens of marine origin (Ikegami et al., 1991). 

 Murine monoclonal antibodies were employed by 

 Miller et al. ( 1991 ) to distinguish three barnacle spe- 

 cies of similar size, and Beck et al. (1992) investi- 

 gated surface antigens of rainbow trout sperm with 

 monoclonal antibodies. 



Immunological detection methods with monoclonal 

 antibodies have also been valuable for many aspects 

 of seafood science. Recently, Huang et al. ( 1995) were 

 able to distinguish the commercially valued red snap- 

 per, L. campechanus, from less valuable substitutes 

 by using ELISA with two monoclonal antibodies 

 raised to a red snapper protein. 



It is now evident that identification of species of 

 the western Atlantic snapper at early life history 

 stages by examining external features alone is in- 

 sufficient and that new methods are necessary to 

 increase our knowledge. The use of highly purified 

 lutjanid proteins, together with the production of 

 polyclonal and monoclonal antibodies for a variety 

 of immunoassays, has wide versatility and applica- 

 bility for future studies. 



Literature cited 



An, H., P. A. Klein, K.-j Kao, M. R. Marshall, W. S. Otwell, 

 and C.-i Wei. 



1990. Development of monoclonal antibody for rock shrimp 

 identification using enzyme-linked immunosorbent assay. 

 J. Agric. Food Chem. 38:2094-2100. 

 Arnold, P. I., J. E. Serafy, M. E. Clarke, and D. R. Schultz. 

 In press. An immunological study of predation on hatch- 

 ery-reared, juvenile red drum (Sciaenops ocellatus): de- 

 scription of an ELISA, and predator-prey studies in 

 nature. J. Exp. Mar. Biol. Ecol. 

 Avrameus, S., and T. Ternynck. 



1969. The cross-linking of proteins with glutaraldehyde and 

 its use for the preparation of immunoadsorbents. Immun- 

 ochem. 6:53-66. 

 Barclay, S. L., and A. M. Smith. 



1986. Rapid isolation of monoclonal antibodies specific for 



