Technicon Instruments Corporation

L'association Internationale Abut Scientifique, Dite: 'institut

Cesar L Cambiaso

Pierre L Masson

Joseph Limet

Claude H Moussebois

Adrian E Leek

Floris De Steenwinkel

Jean-Pierre Vaerman

A method for immunological assay of IgM antibodies against at least one said antigen in a liquid sample containing at least IgM and IgG antibodies, such as a biological fluid, comprising reaction of said liquid sample and of antibodies against IgG, and addition to the so obtained reaction product of the said antigen bound to finely divided particles and of a free said antigen, the amount of IgM being inversely proportional to the amount of unagglutinated finely divided particles.

Method for immunological assay of antibodies of various types in a

Immunoassays for antigens or haptens are effected using, instead of an immunoglobulin antibody, the F(ab').sub.2 fragments thereof. In this way, interference from endogenous RF and Clq in the fluid under assay is avoided without the necessity of pre-treating the fluid to remove or inactivate the RF or Clq. The assays may be of various types including latex agglutination and competitive binding assays. A reagent for such use comprising F(ab').sub.2 fragments insolubilized on a water-insoluble substrate, especially a particulate substrate, is described.

Immunoassays using F(AB').sub.2 fragments

A liquid, particularly but not exclusively a liquid of human origin such as blood serum or urine, is immunoassayed for antibodies, antigens or antibody: antigen complexes, using as a reagent in the analysis an active fraction from mouse ascitic fluid. This active fraction is a euglobulin and has the ability, like human C1q, to combine with antibody:antigen complexes but not with free antibody or antigen. Unlike human C1q, however, it remains active at high pH's and its activity is not destroyed by 0.1 M putrescine or 0.1 M hydrazine, so analyses on human body fluids can be carried out at high pH's or in the presence of putrescine and hydrazine, without interference from endogenous human C1q. The active fraction is a very broadly applicable reagent in immunoassays and particularly useful in techniques involving agglutination of latex particles.

Immunoassay using ascitic fluid

A particle agglutination assay for antigens, antibodies and other binding proteins such as rheumatoid factor, uses two different, microscopic or submicroscopic particulate reagents. The first particulate reagent binds with the antigen or antibody under assay, and then the second particulate reagent is added which binds only to those first reagent particles which have bound to the antigen or antibody under assay, so causing agglutination. The free unbound first or second particles are assayed to indicate the presence and/or amount of the antigen or antibody under assay. The assay of rheumatoid factor may be so conducted to reveal the fractions of each immunoglobulin class of RF present. The assay is particularly useful for small quantities of antibodies present in human sera indicating allergy, infection or autoimmune diseases.

Iummunoassay involving agglutination

Insolublized antibodies, F(ab').sub.2 fragments thereof and protein antigens are made by coupling to a water-insoluble substrate using a bridging agent which covalently binds to sulphur atoms in antibody, fragment or protein rather than to free amino groups therein. This is preferably effected by using a chloracetyl-terminated bridging agent. Immunoassays utilizing these insolubilized reagents are described. One novel class of bridging agents comprising anhydride and monochloracetyl end groups, which is useful for making the said reagents, is also disclosed.

Insolubilized proteins and immunoassays utilizing them

The presence of antibodies (Ab) and antigens (Ag) in a liquid is detected by mixing the liquid with two different particulate reagents which mutually agglutinate but whose agglutination is inhibited by the particular Ab or Ag under assay. By detecting the extent of agglutination, the presence or absence of the Ab or Ag can be confirmed. By measuring the extent of agglutination or non-agglutination quantitatively, an amount of Ab or Ag can be determined. The particulate reagents preferably comprise latex.

Mixed agglutination

In the analysis of liquids, particularly biological liquids such as serum or urine, mouse serum or a component of mouse serum is used as a reagent. The reagent has the ability to combine with antibody:antigen complexes (but not free antibody or antigen) and to combine with IgG and IgM. It differs markedly from human C1q in that it remains active at high pH and in the presence of certain substances known to destroy human C1q. It is a very broadly applicable reagent in immunoassays, and particularly useful in techniques involving agglutination latex particles.

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Kraainem, Belgium

Cesar L Cambiaso