An Investigation of the Cross Reactivity of Antibodies Against the Immunoglobulins of Several Mammalian Species with Immunoglobulins of the Harbor Seal, Phoca vitulina
In studying the humoral immune system of marine mammals, it is important first to understand the basic characteristics of their immunoglobulins, including methods of isolation, structural and chemical properties, and normal levels of those immunoglobulins in physiological fluids such as serum. Since the study of marine mammal immunology is still relatively in its infancy, commercial reagents to use in diagnostic tests are not yet available. However, advantage can be taken of homologies between the immunoglobulins of different species until monoclonal antibodies specific for isotypes o these animals can be produced.
Immunoglobulin isotypes IgG, IgA, and IgM, of the harbor seal, Phoca vitulina, were tested for cross reactivity with reagents created against the same isotypes of other species, in order to permit formulation of an assay using these reagents to determine normal levels of these immunoglobulins in serum. Initially, harbor seal serum was subjected to affinity chromatography methods that have been employed successfully in isolating immunoglobulins of several other mammalian species. IgG, IgA, and IgM were isolated by these methods and subjected to SDS-PAGE. Presumptive identity of harbor isotypes was confirmed by comparison with bands generated by dog isotypes run on the same gel as controls. Sigma Gel analysis showed the molecular weights of heavy chains and light chains of all three immunoglobulins to be similar to those of other mammalian species. Gamma heavy chain was found to be approximately 57,386 Daltons with the light chain at 27,689 Daltons. Alpha heavy chain was 60,777 Daltons and the light chain was 28,587 Daltons. Mu heavy chain was 82,226 Daltons with the light chain being 28,746 Daltons. Antibodies produced against immunoglobulins of other species, including the dog and human, were tested for cross reactivity with harbor seal immunoglobulins. Anti-human reagents were used previously to determine Ig levels in the milk of elephant seals.1 In order to determine if anti-human reagents or anti-dog reagents could be used in a similar way with harbor seals, methods including enzyme-linked immunosorbant assay (ELISA), Western blot, and Grabar Williams immunoelectrophoresis were used. Initial tests show that harbor seal IgG cross reacts with anti-dog IgG, IgA, and IgM as well as anti-human IgG and IgM. There is a slight cross reactivity between harbor seal IgA and anti-dog IgM. Harbor seal IgM reacts with anti-dog IgM as well as anti-human IgG, IgA, and IgM. The conclusion is that these cross-reactive antibodies can be employed in quantitative immunoassays for harbor seal isotypes.
1. Marquez MEI, Carlini AR, Baroni AV, Slobodianik NH, Ronayne de Ferrer PA, Godoy MF. 2000. Shifts in immunoglobulin (IgG, IgM, and IgA) levels in the milk of southern elephant seals, at Potter Peninsula, King George Island, Antarctica. Polar Biology 26: 151-156.