Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-06-25
2001-07-10
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007240, C435S007500, C436S506000, C436S513000, C436S538000, C436S541000
Reexamination Certificate
active
06258549
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a test system by ELISA (enzyme-linked immunosorbent assay). It concerns an ELISA system to detect biochemical entities in very minute amounts and in the presence of structurally very similar compounds, which are differentiated by means of monospecific antibodies, such as immunoglobulins of various species.
DESCRIPTION OF THE RELATED ART
In conventional ELISA systems either the antigen which is to be detected, or a specific antibody which binds to the respective antigen, is bound to a solid phase (microtiter plate) by hydrophobic interactions: the protein interacts with the solid phase, usually a polystyrene surface at high pH. Although this bond is responsible for all the consecutive steps in the procedure, it remains the weakest bridge to the assay support, the ELISA plate. Strong detergents at higher concentrations such as 0.05% are able to diminish the amount of reagent bound to the plate and can even abolish binding totally. The consecutive steps in ELISA technique, such as the binding of an antigen to the solid phase-bound antibody and further binding of a second antibody, occur with an affinity of approximately 10
−12
to 10
−10
mol per liter. One can view this as being similar to an inverse binding cascade from the bottom of the plate to the top, as with a pyramide standing on its top.
Another concern is the intramolecular event upon binding. A protein such as a cell-receptor or an antibody behaves very flexibly according to its polypeptide structure, which forms, a complex architecture in solution. This explains its high specificity and selectivity to the ligands which are to bind to it in vivo. Enzymatic activities for example are entirely dependent upon the proper formation of the active site pocket, which itself remains flexible in order to engulf the substrate and release the product. Cell-receptors such as Fc&egr;RI&agr; react with an affinity for IgE in vivo of 10
−10
mol per liter provided the respective active site is properly exposed. Most proteins react to binding to a given surface with a dramatic change of their tertiary structure, i.e. they unfold, refold, hide their active site or change their conformation in such a way that their reactivity toward a given ligand is altered or even cancelled. In order to circumvent this disadvantage, in conventional ELISA systems a catching antibody is used. This antibody binds to the polystyrene plate and exposes the high affinity hyper-variable region toward the incoming antigen. The antigen is then detected by a second antibody, which is labelled directly or indirectly (via biotin/avidin) with an enzyme. This enzyme will cleave a chromogenic substrate, which itself is converted from the leucoform to the chromoform and thus visualizes the presence of the antigen in question. But even catching antibodies may affect a given protein by changing its conformation. This is demonstrated by many examples of therapeutic antibodies whose mode of action is the blocking of an active site on, or the alteration of, a biospecific molecule.
SUMMARY OF THE INVENTION
The present invention concerns an assay for high capacity screening of substances interfering with the attachment of human IgE to its high affinity receptor Fc&egr;RI&agr;, and/or of substances capable of detaching already bound IgE from this receptor, which is devoid of the above disadvantages. It comprises reacting a solution of a biotinylated Fc&egr;RI&agr; receptor with IgE, transferring the resultant binding reaction mixture to a streptavidin-coated plate and quantifying by means of an appropriate enzyme-labeled antibody. More specifically, it comprises reacting biotinylated IgE-receptor with IgE in the presence or absence of interfering or inhibitory substances, such as non-biotinylated IgE-receptor at well-defined molar concentrations, temperature, pH and salt conditions, thereby eliminating the structural influence of solid phases, and transferring after a defined incubation time an aliquot of the binding reaction mixture to a streptavidin-coated multiple well plate, where the biotinylated IgE-receptor/IgE complex is quantified by means of an appropriate enzyme-labeled antibody, such as horse radish peroxidase (POD)-labeled antibody against human IgE.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The principle of this assay method is as follows: biotinylated IgE-receptor is first reacted with IgE in the presence or absence of interfering or inhibitory substances, such as non-biotinylated IgE-receptor, at well-defined molar concentrations, temperature, pH and salt conditions, thereby eliminating the structural influence of solid phases. After a defined incubation time an aliquot of the binding reaction mixture is transferred to a streptavidin-coated multiple well plate, where the biotinylated IgE-receptor/IgE complex is trapped and quantified by means of an appropriate enzyme-labeled antibody, such as horse radish peroxidase (POD)-labeled antibody against human IgE.
During the reaction of the receptor with its ligand (IgE) in solution, both molecules float freely in solution and thus are not influenced by the structural modification of solid phases. They receive their conformation by the given pH and salt concentration and exert their maximal binding affinity. After a given incubation time, selected according to the results of appropriate equilibrium measurements, the receptor/ligand complex has to be deprived of all other compounds. The receptor is therefore biotinylated prior to the above reaction and an aliquot of the reaction is transferred to a streptavidin-coated multiple well plate. In order to build up an affinity cascade from the bottom of the multiple well plate to the top of the reactants, the binding of streptavidin with the plate surface is preferably made covalent. For example, a microtiter plate (such as a DNA-Bind plate) chemically modified with e.g. a N-oxysuccinimide ester coating is used, which reacts with nucleophiles such as primary amines under formation of a covalent bond. This bond is stronger than 10
−15
moles per liter. For the next step, the biotinylation of the receptor, a chemically modified biotin, such as NHS-LC-Biotin is used, which comprises an extended spacer arm of approximately 22.4 Å in length. This long chain analogue reduces steric hindrances associated with the binding of four biotinylated molecules on one streptavidin molecule. The target of biotinylation on the IgE-receptor molecule is thereby well defined and interference with the ligand is avoided. The binding affinity of biotin to streptavidin is also known to be 10
−15
moles per liter. Thereby the first two steps of the binding cascade are established and reach from the bottom of the plate to streptavidin to the biotinylated receptor. The affinity constants are decreasing from the bottom to the top. At this stage the reactants, which have found their partners in solution under appropriate conditions, are trapped by the means of the streptavidin plate, and the bound IgE is then detected with an appropriate enzyme-labeled antibody, such as POD-labeled antibody against human IgE. The affinity cascade now reaches from <10
−15
to 10
−15
to 10
−10
to 10
−12
for the anti-IgE antibody toward IgE. Any interference of biochemical material and especially of IgE-receptor binding inhibitors will occur at the point with the lowest affinity, which resides between the IgE and the receptor (10
−10
), and not at the point of attachment of the assay to the plate. This would be read as a false positive result—an artifact of the system.
The above provides the basis for the detection of biomolecules other than IgE but which are also binding to Fc&egr;RI&agr;. The prerequisite therefor is the abolishment of the binding of IgE to the receptor in order to keep the receptor site free for other molecules. This is achieved by means of an anti-IgE antibody as BSW17 which binds to the receptor-binding site of the IgE and thus renders the molecule non-effective, whereby even in the pr
Auer Manfred
Hammerschmid Franz
Stingl Georg
Ferraro Gregory D.
Novartis AG
Saunders David
Tung Mary Beth
Wildman David E.
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