Immunoassay for anti-dirofilaria immitis antibody

A method for purifying soluble non-cross-reactive antigenic components of Dirofilaria immitis from a crude extract of Dirofilaria immitis, the soluble non-cross-reactive antigenic components being non-cross-reactive with anti-Toxocara canis antibody, the crude extract including cross-reactive subcomponents which are cross-reactive with both anti-Dirofilaria immitis antibody and anti-Toxocara canis antibody, the method comprising the following steps:

• step (a):
• incubating the crude extract of Dirofilaria immitis with a solid phase immunoadsorbent having anti-Toxocara canis antibody attached thereto for binding the cross-reactive subcomponents, and then
• step (b):
• eluting and collecting the unbound soluble non-cross-reactive antigenic components of the crude extract from the solid phase immunoadsorbant without eluting the bound cross-reactive subcomponents,
• whereby the collected soluble non-cross-reactive antigenic component is substantially free of the cross-reactive antigenic subcomponent.

In an immunoassay reagent for use in an antibody assay for detecting anti-Dirofilaria immitis antibody in a serum sample, the immunoassay reagent including:

• a solid phase carrier and
• soluble antigenic components of anti-Dirofilaria immitis,
• said antigenic components being attached to said solid phase carrier for binding and immobilizing anti-Dirofilaria immitis antibody,
• wherein an improvement enables the antibody assay to discriminate between the detection of anti-Dirofilaria immitis antibody and the detection of anti-Toxocara canis antibody, the improvement comprising:
• said soluble antigenic components consisting essentially of non-cross-reactive antigenic subcomponents which are reactive with anti-Dirofilaria immitis antibody and non-cross-reactive with anti-Toxocara canis antibody.

In an immunoassay reagent for use in an antibody assay for detecting anti-Dirofilaria immitis antibody in a serum sample, the immunoassay reagent including:

• an agglutinizing reagent and
• soluble antigenic components of anti-Dirofilaria immitis,
• said antigenic components being attached to said agglutinizing reagent for binding and agglutinating anti- Dirofilaria immitis antibody,
• wherein an improvement enables the antibody assay to discriminate between the detection of anti-Dirofilaria immitis antibody and the detection of anti-Toxocara canis antibody, the improvement comprising:
• said soluble antigenic components consisting essentially of non-cross-reactive antigenic subcomponents which are reactive with anti-Dirofilaria immitis antibody and non-cross-reactive with anti-Toxocara canis antibody.

A method for purifying a crude extract of Dirofilaria immitis to obtain soluble antigenic components which are reactive with anti-Dirofilaria immitis antibody and which are non-cross-reactive with anti-Toxocara canis antibody, the method comprising the following steps:

• step (a):
• combining and incubating the crude extract of Dirofilaria immitis with a solid phase immunoadsorbent including an anti-Dirofilaria immitis monoclonal antibody attached thereto, the anti-Dirofilaria immitis monoclonal antibody having a binding specificity for soluble antigenic components which are non-cross-reactive with anti-Toxocara canis antibody, then
• step (b):
• washing the immunoadsorbent to remove unbound components of the crude extract, then
• step (c):
• eluting the bound soluble antigenic components from the immunoadsorbent by rinsing with a releasing agent, and then
• step (d):
• collecting the eluted soluble antigenic components and neutralizing the collected releasing agent, the eluted soluble antigenic components being reactive with anti-Dirofilaria immitis antibody and non-cross-reactive with anti-Toxocara canis antibody