Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-08
2003-11-11
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007900, C435S007920, C435S007940, C435S965000, C436S512000, C436S513000, C436S518000, C530S866000
Reexamination Certificate
active
06645732
ABSTRACT:
The invention concerns a method for the determination of antigen-specific antibodies of the immunoglobulin G class in body fluids by incubating the sample with at least two different receptors R
1
and R
2
wherein both receptors are capable of binding specifically to the antibody, R
1
is bound or can be bound to a solid phase and R
2
carries a label, separating the solid from the liquid phase and measuring the label wherein a conjugate of a binding partner in monomeric form which is specifically recognized by the antibody to be determined and a label is used as R
2
.
In particular the invention concerns a method for the specific detection of immunoglobulins of the IgG class in the presence of immunoglobulins of the IgM class.
In response to the introduction of foreign substances the immune system of a mammalian organism produces antibodies which are also called immunoglobulins. They defend against foreign substances which are also referred to as antigens. The immunoglobulins can be divided into five different classes. One distinguishes between immunoglobulins of the M, G, A, E and D classes. Each of these five immunoglobulin classes differ in the composition of the heavy chain which is referred to as the &mgr;, &ggr;, &agr;, &egr; and &dgr; chain.
Each immunoglobulin class has a different function in the organism. Immunoglobulins of the M class appear with the first contact with the antigen the so-called first immunization. However, the concentration of these immunoglobulins decreases rapidly as the infection progresses. The immunoglobulins of the G class are firstly slowly formed after a first immunization and occur in large quantities when there is a second infection with the same antigen. The immunoglobulins of the A class are found on the mucous membrane surfaces of the organism and are responsible for the defence processes there. The immunoglobulins of the E class are mainly responsible for allergic reactions. The exact function of immunoglobulins of the D class is hitherto unknown.
The individual immunoglobulin classes occur in very different concentrations in the blood. Thus immunoglobulins of the G class (IgG) are the major class in normal human serum with a share of about 75% that corresponds to a serum content of 8 to 18 mg/ml. The second most frequently occurring immunoglobulin is IgA which has an average serum concentration of 0.9 to 4.5 mg/ml. Immunoglobulins of the M class are present at a concentration of 0.6 to 2.8 mg/ml, immunoglobulins of the D class are present at a concentration of 0.003 to 0.4 mg/ml. The proportion of IgE antibodies is lowest which only occur at a concentration of 0.02 to 0.05 &mgr;g/ml in serum.
For the differential diagnosis of many diseases it is important to detect antibodies of one or several quite particular immunoglobulin classes which are specific for a particular antigen. A satisfactory diagnosis of viral, bacterial and parasitic infections can only be ensured by means of a class-specific antibody test or by excluding the presence of certain immunoglobulin classes (e.g. detection of IgG and IgA antibodies but no detection of IgM antibodies). This is particularly important for the differentiation between fresh or acute infections and infections that have occurred earlier as well as for the clinical monitoring of the course of an infection. The class-specific detection of antibodies is especially important for HIV, hepatitis A, hepatitis B, toxoplasmosis, rubella and chlamydia infections. The class-specific detection of antibodies specific for a particular antigen is also necessary for the determination of the titre of protecting antibodies and to check the success of an immunization. Hence from a diagnostic view point there is great interest in the detection especially of antibodies of the non-acute stages of infections such IgG and IgA antibodies.
Various methods have been described in the state of the art for detecting antibodies of a particular class that are specific for an antigen. Hence antigen-specific antibodies of a particular class are frequently detected by binding the specific antibodies to a solid phase coated with the specific antigen. The immunoglobulins (Ig) specific for the antigen which are now bound to the solid phase are detected by binding antibodies which are specifically directed towards human Ig of a certain class to the Ig molecules to be detected. The antibodies directed towards human Ig are provided with a label by means of which the detection takes place. However, such a test procedure is only possible if all unspecific non-bound Ig is removed by washing before the reaction with the class-specific labelled antibodies directed towards human Ig. Thus a one-step test procedure as is often required for automated systems is not possible.
According to the method described in the U.S. Pat. No. 4,292,403 antigen-specific antibodies of a particular immunoglobulin class are detected by immobilizing a class-specific antibody on a solid phase which binds sample antibodies to be determined, subsequently adding the specific antigen and binding the bound antigen to a further labelled antibody that is specific for the antigen. However, a disadvantage of this method is that all antibodies of the class to be determined must bind to the class-specific immobilized antibody. The sample antibodies are not bound antigen-specifically. This could impair the sensitivity of the test since there may not be sufficient free binding sites for the antigen-specific antibody. Several wash steps are also necessary in this test procedure. This method does not enable a one-step test procedure.
One possibility of carrying out an antibody detection in a one-step test is provided by the so-called bridge test. The bridge test concept is described in EP-A-0 280 211. In this method a first receptor which is capable of specific binding to the antibody to be determined is bound to a solid phase such as for example an antigen. The antibody to be determined binds to the solid phase-bound antigen. In addition a further specific antigen is present in the test mixture which is provided with a label. The antibody is detected by means of the label. In this test all antigen-specific antibodies are detected and not only the antibodies of a particular class.
In EP-A-0 307 149 and in the U.S. Pat. No. 5,254,458 methods based on the bridge test principle are disclosed for the detection of antibodies which are directed specifically towards an antigen. In this case peptides produced recombinantly which are derived from a certain epitope of the antigen are used to bind the antibody to be detected. A peptide is immobilized to a solid phase. The sample antibody binds to the peptide. A further labelled peptide is bound to the sample antibody for the detection. The recombinant peptides are expressed in different organisms in order to increase the specificity of the test. Also in this method antibodies of all classes bind to the peptides. It is not possible to for example differentiate between IgG and IgM antibodies.
EP-A-0 386 713 describes a method for the detection of antibodies against HIV using two solid carriers in which different HIV antigens are immobilized on both solid carriers which are each contacted with an aliquot of a sample and a labelled HIV antigen wherein the presence of antibodies is detected by a positive reaction in at least one of the tests. Polypeptides produced recombinantly are disclosed as HIV antigens. A method based on Western blot is disclosed in EP-A-0 627 625 in which HIV antibodies can also be detected by means of recombinant proteins or synthetic peptides. However, both methods do not allow the class-specific detection of antigen-specific antibodies.
The previous methods do not enable an antigen-specific antibody of a certain immunoglobulin class to be detected in a one-step method. The immunological methods of detection known from the state of the art based on the bridge test concept in which a labelled antigen and an antigen capable of binding to a solid phase are used, do indeed enable a one-step test. However, up to now it has
Faatz Elke
Schmitt Urban
Amick Marilyn L.
Nguyen Bao-Thuy L.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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