Development of techniques for observing variability at the major histocompatibility complex (MHC) of fishes could prove an important first step in understanding the genetic bases of disease resistance. In this study, using goldfish (Carassius auratus) as a model system, three approaches to generating antisera to putative MHC molecules and two methods for detecting antibody reactivities were evaluated.

Seven full-sib families were produced, and red blood cells (RBCs) of goldfish family members were screened for reactivity with a panel of absorbed antisera. The antisera panel consisted of fish anti-fish, chicken anti-chicken, and chicken anti-fish antisera. The fish anti-fish antisera was produced by injecting RBCs from each parent into its mate, and the chicken anti-fish antisera was produced by injecting parental goldfish RBCs into chickens. The chicken anti-chicken antisera were obtained from a genetics laboratory where MHC-specific antisera had been prepared previously. The pattern of presence or absence of agglutination upon mixing with the respective reagents in this panel of antisera was regarded as the phenotype of the individual tested.

Agglutinations observed macroscopically or microscopically were easily scored as positive or negative. Particular phenotypes were observed among individuals both within and between families. The large numbers of phenotypes observed may indicate: (1) the need for additional absorptions in the preparation of antisera, or (2) segregation of additional sets of phenotypic MHC haplotypes in the tetraploid goldfish. The utility of chicken anti-chicken reagents in serotyping of fish was demonstrated.

Use of the traditional approach to conducting hemagglutination assays limited the number of assays executed because of the amount of blood required. In order to minimize the sample volumes required, antibody reactivities were evaluated by flow cytometry employing appropriate fluorescein labeled antibodies. Using this approach, scoring of positive and negative results was equivocal, and results did not always agree with those scored by hemagglutination assays.

Results of this study strongly suggest that the development of immune allo- and xeno-antisera and use of hemagglutination assays can be used to characterize genetic variability of the MHC of fishes. Understanding of immunogenetic variability in fishes could be used to develop strains resistant to economically important fish pathogens.