Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-15
2002-12-03
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, C435S962000, C436S507000, C436S509000, C436S512000, C436S513000, C436S518000, C436S825000
Reexamination Certificate
active
06489129
ABSTRACT:
The invention concerns a method for the determination of antigen-specific antibodies of the immunoglobulin M class in body fluids by incubating the sample with at least two different receptors R
1
and R
2
where 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, wherein an essential component of R
1
and optionally also of R
2
is a binding partner in polymeric form which is specifically recognized by the antibody to be determined and binding partners of the same specificity are used in monomeric form to reduce interference by IgG antibodies.
In particular the invention concerns a method for the specific detection of immunoglobulins of the IgM class in the presence of immunoglobulins of the IgG class and interfering factors such as rheumatoid factors.
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 after the first contact with the antigen, the so-called primary 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 primary 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 and they 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 distinguishing 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 when determining the titre of protecting antibodies and to check the success of an immunization. For the diagnosis of fresh, acute infections it is of particular interest to detect antibodies of the IgM class which are specific for an antigen. However, various interfering factors such as for example the presence of IgG antibodies of the same specificity frequently interfere with the detection of antigen-specific IgM 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. In addition antibodies of all classes which are specific for the antigen bind to the solid phase in the first step. If the antigen coating of the solid phase is not high enough, competing reactions of the various antibody classes for binding to the antigen can occur. This can impair the sensitivity of the test.
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 such as for example an antigen which is capable of specific binding to the antibody to be determined is bound to a solid phase. 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. However, in this test all antigen-specific antibodies are detected and not only the antibodies of a particular class.
An additional interference when determining antigen-specific IgM antibodies is caused by rheumatoid factors. Rheumatoid factors are themselves usually antibodies of the IgM class which generally have a high affinity for the Fc regions of IgG antibodies. As a result rheumatoid factors seemingly present IgG antibodies so that the rheumatoid factors are bound in an immunoassay for specific IgM antibodies. If the rheumatoid factors have bound IgG molecules with the specificity that is to be detected, this can result in false-positive measurement results.
This problem in the class-specific detection of antigen-specific antibodies is the subject matter of DE 33 03 793. This describes a method for the detection. of an antigen-specific antibody of a certain Ig class (“IgX”) in which interference by rheumatoid factors is eliminated by adding anti-IgG antibodies. In the method the specific antigen, such as a virus antigen, is applied to a solid carrier. The virus antigen bound to the solid phase is contacted with the sample. In the next step the unbound sample is removed and the solid phase-bound complex of antigen-IgX is detected with an anti-IgX antibody. In order to avoid interference by rheumatoid factors especially in IgM tests, the sample is treated with anti-IgG antibodies before the test. The IgG antibodies complexed in this manner are thus no longer available for attack by rheumatoid factors so that the rheumatoid factors are not able to bind antigen-specific IgG molecules which could in turn result in false-positive results. However, all IgG antibodies are bound regardless of their specificity. The precipitation of interfering IgG antibodies with anti-IgG antibodies can lead to undesired precipitates and turbidity which can have an adverse effect on the entire test. In addition sample pretreatment with anti-IgG antibodies is complicated.
A further method of eliminating interference by antibodies of other classes with the same specificity is disclosed in WO 96/14337. In this case antibodies or antibody fragments which react specifically w
Faatz Elke
Ofenloch-Hahnle Beatus
Schmitt Urban
Amick Marilyn L.
Nguyen Bao-Thuy L.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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