Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-24
2002-04-30
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06379909
ABSTRACT:
PRIOR ART
WO 94/11734 describes a two-site immunoassay for an antibody using a chemiluminescent label and a biotin bound ligand, said method comprising the steps of (a) mixing the liquid sample with a ligand antigen, antibody or hapten bound to biotin or a functional derivative thereof, an antibody directed against the antibody to be detected bound to paramagnetic particles and a chemiluminescent acridinium compound bound to avidin, streptavidin or a functional derivative thereof to form a solid phase complex, (b) magnetically separating the solid chase from the liquid phase, (c) initiating a chemiluminescent reaction, if any, in the separated solid phase and (d) analysing the separated solid phase for the presence of a chemiluminescent phase, which is indicative of the presence of said antibody in the sample.
The prior art method is particularly suitable for measuring the concentration of specific immunoglobulins in body fluids, such as a specific immunoglobulin selected from the group of IgA, IgD, IgE, IgG, IgM and subclasses thereof.
The prior art method is also suitable for the detection and quantification of the total content of immunoglobulins in a class or subclass, such as IgA, IgD, IgE, IgG, IgM and subclasses thereof.
In a preferred embodiment of the prior art method the formation of the solid phase complex is effected in two steps, viz. a first step wherein the sample is mixed with the biotin bound ligand antigen, antibody or hapten and the antibody bound to paramagnetic particles so as to form a first solid phase complex, and a second step wherein the chemiluminescent acridinium compound is added to the first solid phase complex to form a second solid phase complex.
However, practical use of the prior art method has revealed the fact that in some applications of the method interference from other types of immunoglobulins and/or from other types of immunologically active serum components than the one to be measured occurs leading to errors in the results obtained.
The article “Capture assay for specific IgE”, V. Olivieri et al, Journal of Immunological Methods, 157 (1993) 65-72 discloses an assay for the measurement of specific IgE in the serum of allergic patients using monoclonal anti-human IgE (coated to the wells of a microtiter plate) and biotinylated allergens in solution. In a single incubation IgE is bound to the solid phase through the Fc fragment and biotinylated allergens react with their specific IgE Fab regions. In a second step, streptavidin-horseradish peroxidase conjugate is added to reveal the amount of biotin fixed on the solid phase. The article studies the interference from high levels of non-specific IgE and from allergen-specific IgG. The assay is found to be unaffected by allergen-specific IgG.
WO 98/16829 discloses an assay, wherein an anti-immnunoglobulin is coupled to a microtiter plate well, which is then washed. The test serum is then added to the well to capture all of total targeted immunoglobulin in the test sample. Then, the biological fluid sample is aspirated, and the microtiter plate is washed. The captured antibody on the microtiter plate is then exposed to biotinylated antigen, the plate is washed, and streptavidin/alkaline phosphatase conjugate is added, and the plate is washed again. The prior art assay may be used to monitor the effect of treatment on
H. Pylori
infection with standard antibiotic therapy.
SUMMARY OF THE INVENTION
A first object of the invention is to provide a method of the type, wherein the targeted antibody is complexed to a Fc directed antibody coupled to a solid particle and to a Fab directed ligand, which does not suffer from the above explained drawback of interference from other components of the test sample.
This first object is achieved with the method of the invention, the essential new feature of the invention being that an additional sequence of separation and washing of the intermediate solid phase complex consisting of particle with reactant antibody and sample antibody is carried out prior to addition of ligand.
The method of the invention is based on the recognition that by introducing such an additional sequence of separation and washing, potentially interfering excess material from the liquid sample as well as potentially interfering excess component (ii) may be removed from the method thus eliminating the risk of interference of the said factors in subsequent steps. It has surprisingly been found that the additional sequence of separation and washing has reduced substantially and in some circumstances eliminated the technical problem of interference between different types of immunologically active serum components.
The reduction of the interference obtained involves a number of technical advantages. In particular, it allows a more precise measurement of problematic sera having a difficult and unpredictable ratio of mixture of antibodies. Furthermore, falsely positive identifications of an antibody may be avoided. Also, the reduction of interference allows precise measurements to be made in a wider antibody concentration range than with prior art methods.
A second object of the invention is to provide a method, which is capable of evaluating and/or predicting the effect of a Specific Allergy Vaccination (SAV).
This second object is obtained by the nature and the temporal development of the interference between different types of immunologically active serum components, e.g. antibodies, are used as parameters for evaluating/predicting the effect of a Specific Allergy Vaccination treatment. Thus, it has surprisingly been found that the said parameters hold valuable information about the immunological status of a person as well as the response of a person to a selected treatment scheme.
A third object of the invention is to provide a method of evaluating the immunological status of a subject.
The third object of the invention is obtained by the nature and the temporal development of the interference between different types of immunologically active serum components, e.g. antibodies, are used as a parameter for evaluating the immunological status of a subject, in particular evaluating/predicting the effect of allergy treatment, allergy vaccination treatment or Specific Allergy Vaccination treatment. Thus, it has surprisingly been found that the said parameter hold valuable information about the immunological status of a person as well as the response of a person to a selected treatment scheme.
A fourth object or the invention is to provide a method of evaluating the effect of allergy treatment of a subject.
The fourth object of the invention is obtained based on the recognition that the measurement obtained with the subassays 1, A and C, i.e. a measurement, which is carried out in the presence of interfering factors in the sample, is particular useful for evaluating the effect of allergy treatment, allergy vaccination treatment and Specific Allergy Treatment (SAV). Thus, the measurement obtained with this method is obtained under conditions, which correspond to in vivo conditions, and hence is a more physiological and clinical relevant measurement for evaluating treatment effects.
First Aspect of the Invention
A preferred embodiment of the first aspect of the invention is characterized in that component (iii) of step (r′), (r) or (r″) and component (iv) of step (s′), (s) or (s″), respectively, are added in one operation.
A first alternative embodiment of the first aspect of the invention is characterized in that the three-component solid phase complex formed in step (r′), (r) or (r″) prior to subjecting it to step (s′), (s) or (s″), respectively, is washed to remove non-complex bound compounds.
Second Aspect of the Invention
Specific allergy vaccination (SAV), formerly known as specific Immunotheraphy or Hyposensitization, has been used for the treatment of Type 1 IgE mediated allergic disease since the beginning of this century.
The general benefits obtained through SAV are: a) reduction of allergic symptoms and medicine consumption, b) improved
Beck Tine Charlotte
Bogestrand Soren
Ipsen Hans-Henrik
Johansen Niels
Morkeberg R Ikke
Alk-Abello A/S
Le Long V.
Padmanabhan Kartic
Watov & Kipnes P.C.
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