Acylated protein aggregates and their use as signal enhancers in

Details Acylated protein aggregates and their use as signal enhancers in Acylated protein aggregates and their use as signal enhancers in

1996-02-29

1999-12-07

Achutamurthy, Ponnathapura

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 792, 435 72, 435 71, 530363, 530362, G01N 3353

Patent

active

059981571

DESCRIPTION:

BRIEF SUMMARY
The invention addresses acylated protein aggregates, their manufacture, and their use in an agent and in a reagent for immunological assays, and their use as signal enhancers in immunoassays for the detection of antibodies and a corresponding immunological detection method.
Immunological detection methods have gained great importance over the last years. This serves to detect the presence of drugs, hormones, proteins, and especially infectious organisms in biological samples in a rapid and exact manner. In all immunological detection methods, there is a specific binding reaction between a first specific binding partner, the substance to be detected (analyte) and a second specific binding partner which specifically reacts with the ligand and binds it. Ligand and specific ligand-binding partner form a specific binding pair, generally a complex between an antigen and an antibody or antibody fragment. It is possible that more than one ligand or one binding partner react with each other in each reaction. These specific binding reactions are detected in various ways. Generally, one participant in the specific binding reaction is labeled. Conventional labeling methods make use of radio-isotopes, chromogens, fluorogens, or enzymatic labels. In heterogeneous immunoassays, one of the binding partners is immobilized on a solid phase.
The detection of specific antibodies that are directed against an antigen in an heterogeneous immunoassay, especially so-called sandwich immunoassays, is generally accomplished such that in a first incubation step, a solid phase bound antigen is brought into contact with the sample to be assayed, usually human serum or plasma. During incubation, the antibodies to the antigen contained in the sample bind to the wall-bound antigen. After the reaction, the test mixture is washed and in a second reaction step, the solid phase bound antibodies are detected with the aid of a second antibody that bears a label and is against the antibody class to be determined. This detection is accomplished in that a labeled antibody (monoclonal or polyclonal) directed against the antibody class to be determined or a labeled analyte analog bind in a second incubation step to the antibodies that have bound the wall-bound antigen in the first incubation step. If IgG antibodies are to be detected, it is preferred to use a labeled anti-IgG; if IgM antibodies are used, an anti-IgM antibody is preferred. After the second incubation step, the test mixture is washed and the non-bound, excess labeled anti-antibodies or analyte analogs are removed. In a third step, the amount of bound antibody is detected such that, for example, in an enzyme immunoassay the test mixture reacts with a substrate solution and the resulting coloration is photometrically measured. The absorbance or the signal of this coloration is proportional to the amount of bound antibody.
In a particular embodiment of these antibody immunoassays, the antigen is not directly bound to the solid phase, but is bound to the solid phase before or preferably during the assay via another specific binding partner. In a preferred manner, an antigen to which biotin is covalently linked, binds to a streptavidin-coated solid phase.
A particular type of interference in antibody tests has been noticed especially during the detection of IgG antibodies to an antigen, e.g. HIV, HCV, toxoplasmosis antibodies and the like. If the same amount of a sample of the human serum containing a high concentration of an antibody directed against an antigen (e.g. antibodies to HCV) is diluted with different sera which do not contain an antibody to this antigen, there are significant differences in the recovery of the antibody amount in the so-diluted sera, although all sera contain the same total amount of specific antibodies. In the enzyme immunoassays, the samples diluted with sera produce a signal that was by a factor of 2-10 too low. An explanation for this phenomenon is not known, but it could possibly be that human sera contain substances which interfere with the binding of speci

REFERENCES:
Dorder et al, Chem. Abs., vol. 119, 188678p (Equivalent to WO 93/16,735), 1993.
Medi-Physics, Inc., MPI MAA KIT, Oct. 1990.

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