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



735 



wide versatility and applicability for current and 

 future studies of early life history forms. The chro- 

 matographic purification and characterization of a 

 66 kDa glycoprotein from soluble extracts of Lutjanus 

 griseus (Linnaeus), L.jocu (Schneider), andL. apodus 

 (Walbaum) and the production of a specific polyclonal 

 antiserum have generated evidence that the glyco- 

 protein has both interspecies and species-specific 

 epitopes. Further characterization of this and other 

 lutjanid proteins, together with the production of 

 both polyclonal and monoclonal antibodies for im- 

 munologic assays, may greatly facilitate future identi- 

 fication of lutjanids at any stage of their life history. 



Materials and methods 



Adult lutjanid specimens were obtained from local 

 fishermen and skin divers, and species identification 

 was carried out according to Robins and Ray ( 1986) 

 and Robins et al. (1991). 



Buffers and the methods for preparing soluble sa- 

 line fish extracts are described in Schultz and Clarke 

 (1995). In addition to L. griseus, the 13 species and 

 one hybrid investigated here were the following: L. 

 jocu, L. apodus, L. vivanus (Cuvier), L. campechanus 

 (Poey), L. analis (Cuvier), L. synagris (Linnaeus), L. 

 mahogoni (Cuvier), Ocyurus chrysurus (Bloch), L. 

 buccanella (Cuvier), Pristipomoides aquilonaris 

 (Goode and Bean), Etelis oculata (Valenciennes), L. 

 cyanopterus (Cuvier), Apsilus dentatus (Guichenot), 

 and the hybrid L. synagris x O. chrysurus. Separate 

 extracts were prepared in 300 mL of buffer with ap- 

 proximately 30 g of tissue from gutted legal-size adult 

 fish. The soluble, clear extract was concentrated by 

 ultrafiltration (M 10,000 membrane, Amicon Corp., 

 Danvers, MA) at 4°C until the OD 280 exceeded 10. 

 Also extracted were 1) gutted L. griseus juveniles 

 with a total weight of 0.6 g and 2) L. griseus female 

 gonads with oocytes, weighing approximately 3.5 g. 

 Juveniles were collected from Biscayne Bay, Florida. 



Two different antisera were produced in adult fe- 

 male goats according to the innoculation schedule of 

 Schultz and Clarke (1995). The first was a polyva- 

 lent antiserum (anti-GSE) raised against L. griseus 

 whole fish extract ( 14.1 mg protein/mL). The second 

 antiserum (anti-66 kDa) was produced by using the 

 66 kDa protein (0.32 mg/mL) purified from L. griseus 

 saline extracts (see below). 



The y-globulin fraction of anti-GSE and anti-66 

 kDa sera was isolated by 33% ammonium sulfate 

 (A.S.) precipitation at 25°C (Stelos, 1967) and was 

 further purified as IgG by DEAE-Sephacel (Phar- 

 macia BioTech, Uppsala, Sweden) column chroma- 

 tography in 10 mM NaPO4/50 mM NaCl, pH 7.4. The 



IgG-rich fractions were concentrated by ultrafiltra- 

 tion as above. 



To eliminate background interference when anti- 

 GSE was used as the primary antiserum in Western 

 blots, it was necessary to biotinylate the IgG frac- 

 tion (Bayer and Wilchek, 1980). The biotin-conju- 

 gated anti-GSE IgG was recovered by Sephadex G- 

 25 (Pharmacia BioTech) gel filtration in 5 mM 

 NaPO4/150 mM NaCl, pH 7.5 (PBS) and the biotin- 

 protein ratio was 56 nmol of biotin per nmol of IgG 

 (Green, 1965). 



Soluble extracts of L. mahogoni, L. apodus, and 

 Eucinostomus gula (silver jenny) were insolubilized 

 with glutaraldehyde according to the method of 

 Avrameus and Ternynck (1969), with 10 mg of glut- 

 araldehyde per mg of soluble fish protein. Insolu- 

 bilized E. gula was used previously to remove cross- 

 reactivity with preimmune and immune goat sera and 

 unrelated fish proteins (Schultz and Clarke, 1995). 



The y-globulin fraction of goat anti-66 kDa serum 

 was adsorbed for 24 to 48 h at 4°C with 60 mg of 

 insolubilized E. gula extract per mL y-globulin, 

 followed by 24 mg of insolubilized L. mahogoni ex- 

 tract (the 66 kDa protein was not detected in this 

 species in Western blots). The IgG fraction of the 

 adsorbed anti-66 kDa serum (anti-66 kDa IgG) was 

 isolated by DEAE-Sephacel chromatography (see 

 protocol above). This procedure removed all nonspe- 

 cific activity. 



Another portion of the y-globulin fraction of anti- 

 66 kDa serum was adsorbed with 100 mg of 

 insolubilized L. apodus extract for 24 h per adsorp- 

 tion at 4°C in experiments to distinguish the 66 kDa 

 proteins found in soluble saline extracts of L. griseus, 

 L.jocu, andL. apodus (see Results section). 



The buffers and conditions for ion exchange chro- 

 matography with the Fast Protein Liquid Chroma- 

 tography (FPLC) system (Pharmacia Biotech, 

 Uppsala, Sweden) as described in Schultz and Clarke 

 (1995) were modified to include 10 mM EDTA in the 

 column buffers. The sequence was that of Mono S cat- 

 ion exchanger followed by Mono Q anion exchanger. 



Gel filtration on Sephacryl S-300 Superfine 

 (Pharmacia Biotech) in PBS/10 mM EDTA, pH 7.5, 

 was used to separate high molecular weight contami- 

 nants from the 66 kDa protein. 



During purification procedures, protein concentra- 

 tion was monitored by absorbance at 280 nm. Puri- 

 fied proteins were dialyzed against PBS, pH 7.5, to 

 remove EDTA, and the protein concentration was 

 determined spectrophotometrically as described pre- 

 viously (Arnold et al., in press). Methods for electro- 

 phoresis in 10% (w/v) polyacrylamide gels with or 

 without sodium dodecylsulfate (Laemmli, 1970). for 

 Western blots (Towbin et al., 1979), and the molecu- 



