International Institute of Cellular and Molecular Pathology

Technicon Instruments Corporation

Daniel Collet-Cassart

Pierre L Masson

Floris De Steenwinkel

Carl-Gustaf M Magnusson

Carl G Magnusson

Carl-Gustav M Magnusson

Jean-Claude Mareschal

A biological fluid from a first animal species (e.g. human serum) is assayed for an immunogen therein by mixing with the fluid (Ig-minus-Fc) fragments of an immunoglobulin from a second animal species (e.g. rabbit), the immunoglobulin being immunospecific to the immunogen or another component of the mixture whereby the immunogen can be determined. Interference can occur from reaction between first animal species antibodies and the said fragments, and in the method of the invention this interference is avoided or overcome, preferably by also including in the mixture different (Ig-minus-Fc) immunoglobulin fragments from the second animal species which react with said antibodies but not with said immunogen. These different fragments are preferably aggregated. The method of the invention is particularly applicable to particle agglutination assays.

Method of immunoassay

In a particle agglutination assay for an antigen (Ag), there is included in the mixture a limited amount of a substance which binds univalently with a proportion of the Ag present, that Ag which is so bound being unable then to cause agglutination of the particles. In this way, unusually large concentrations of Ag can be assayed in that a proportion of the Ag is bound to the univalent substance and the particle agglutination assay is in effect conducted on the smaller amount of Ag still remaining free in solution.

Particle agglutination assay of antigens

In the immunoassay of a particular protein in a biological fluid, there is frequently interference in the assay by other proteins present in the fluid, e.g. by complement factors or antibodies in human serum. The interference so caused can be avoided by subjecting the fluid to protein-digestion, using for example an enzyme such as pepsin, as a result of which the particular protein of interest, or a fragment thereof, can be assayed without interference by the other proteins. Also, radioallergosorbent tests for particular IgE antibodies can be improved in sensitivity and accuracy, by subjecting the absorbed IgE to enzymic digestion, and then assaying a fragment thereof.

Immunoassay of proteins

A particle counting assay for haptens (small non-protein monovalent substances having a molecular weight below 1500) comprises mixing a liquid sample (e.g. of human origin) containing the hapten, with finely divided inert particles bearing the same hapten (or a specific analogue thereof), an agglutinator such as RF or C1q, and a measured amount of antibody, which amount is insufficient to cause agglutination of all the particles. The amount of hapten is determinable by measuring the extent of the agglutination.

Particle agglutination immunoassay with agglutinator for determining

In particle agglutination immunoassays for an analyte (e.g. an antigen or antibody) in a liquid sample (e.g. human serum), interferences arise due to non-specific protein-protein interactions and the like. These interferences are reduced or overcome by including in the assay mixture a chaotropic or chaotropic-like agent, in a carefully controlled amount. Such agents include guanidine, guanidinium hydrochloride or thiocyanate, sodium or ammonium thiocyanate, and urea, sodium chloride and ethylenediamine tetraacetic acid.

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Kraainem, Belgium

Daniel Collet-Cassart