Chemistry: analytical and immunological testing – Involving iga – igd – ige – or igm
Reexamination Certificate
1995-05-22
2003-10-14
Nguyen, Bao Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving iga, igd, ige, or igm
C436S512000, C436S518000, C436S523000, C436S538000, C436S548000, C436S820000, C435S007720, C435S007900, C435S007940, C435S962000
Reexamination Certificate
active
06632682
ABSTRACT:
The invention relates to an immunochemical method for the detection and for the determination of antibodies which are specific for a particular antigen and are of one of the immunoglobulin classes. This method is suitable for the highly sensitive and specific detection and for the determination of antibodies of one of the immunoglobulin classes A, M, D or E.
Immunoglobulins are antibodies formed by the immune system of the body against foreign substances (antigens, for example proteins of pathogens, bacterial polysaccharides, serum proteins, tissue proteins or other immunoglobulins). The immunoglobulin molecule is composed of one or more sets of 4 polypeptide chains, two heavy chains each having a molecular weight of about 53,000 daltons and two light chains each of about 22,000 daltons, which are connected by disulfide bridges.
Immunoglobulins are generally assigned to the classes G, A, M, D or E and, correspondingly, called IgG, IgA, IgM, IgD or IgE. These 5 immunoglobulin classes differ in the antigenic determinants of the heavy chain, which are called gamma-, alpha-, mu-, delta- and epsilon-chains; in addition, there are also immunoglobulin subclasses of IgG, IgA and IgM.
Immunoglobulins can be split into fragments which retain the antigen-binding property or into fragments without the antigen-binding property. Examples of antigen-binding fragments are Fab, Fab′ and F(ab′)
2
fragments. Examples of fragments without the antigen-binding property are Fc and Fc′ fragments.
The concentrations of immunoglobulins in normal human serum are (in mg/ml): IgG 8-16, IgA 1.4-4, IgM 0.5-2, IgD 0.0-0.4 and IgE 0.000017-0.00045.
The immunoglobulins present in the highest quantity in human serum are those of the IgG class. Immunoglobulins of the IgM class appear very soon after an infection, for which reason their determination is important for the early diagnosis of an infectious disease or for the diagnosis of an acute infection.
The second most abundant immunoglobulins are of the immunoglobulin class IgA and are the most important secretory antibodies.
Immunoglobulins of classes IgD and IgE can be found in elevated concentration in certain pathological processes; for example IgE has properties which sensitize mast cells and it plays a significant part in the pathogenesis of a number of allergic reactions. IgD antibodies are found in autoimmune diseases.
The determination of antigen-specific immunoglobulins, especially of a particular class, is of special importance for detecting particular diseases caused by parasites, bacteria or viruses, it being possible in this connection to distinguish between acute and resolved infections and, where applicable, to draw conclusions about the prognosis.
A large number of immunological methods is known for the determination of immunoglobulins. Methods for the physical separation of immunoglobulins into classes, for example immunodiffusion, immunoelectrophoresis or density gradient centrifugation, are elaborate, inaccurate and susceptible to interference.
Antigen-specific immunoglobulins can be determined in what is called the direct method by immunoassay techniques; these entail an immune component with binding affinity for the antibody class which is to be determined being coupled to a solid carrier, for example antibodies against the &mgr;-chain of human IgM, and the antigen-specific immunoglobulin fraction being detected either by labeled antigen or as a combination of unlabeled antigen and antigen-specific labeled antibody. The fraction of the labeled immune component which is bound to the solid phase and is directly proportional to the concentration of the antibody which is to be detected is measured.
Used for the labeling are, for example, fluorescent and chromophoric substances or radioactive isotopes, enzymes or particles loaded with immune components, such as erythrocytes or latex particles; it is also possible to use a biological function of the antigen used, for example hemolysis, to indicate that reaction has taken place.
A disadvantage of the methods of the state of the art is that the non-antigen-specific immunoglobulin fraction of any particular immunoglobulin class enters into competition with the antigen-specific fraction for the relevant antibody on the solid phase. This may mean that results differ depending on the ratio of these amounts, even if the antigen-specific antibody fraction remains unchanged (the non-antigen-specific immunoglobulin fraction may in such cases vary by a factor of 5 or more).
It is essential in all the so-called direct and indirect methods which have been described and quoted hitherto that, after reaction (incubation) of the sample with the immune component on the solid carrier and before reaction with the detecting immune component, unbound material is removed by washing. This is why these methods are called “two-step methods”.
Hence, an assay with a ready-to-use carrier-bound component requires at least three reaction steps (sample/second immune component/detection reaction) which are separated from one another by at least 2 washing steps, each reaction step itself requiring a certain reaction time so that the sum thereof gives the total assay time.
The object now was to shorten and simplify the direct assay and to eliminate the competition between non-antigen-specific and antigen-specific immunoglobulins in order to permit reliable quantitative determination of the antigen-specific antibody fraction.
It has now been found, surprisingly, that this is possible by contacting carrier-bound immune component, analyte-containing sample and labeled detecting immune component without washing between addition of the sample and addition of the labeled detecting immune component.
This “one-step method” has become possible after successful elimination of two possible interferences:
In the first place, the effect of antigen-specific IgG antibodies must be eliminated so that the reaction thereof with the antigen, which would interfere with the actual detection method, is now zero or only inconsiderable. This interference is possible in principle because, in the determination of antigen-specific antibodies of one of the immunoglobulin classes IgA, IgM, IgE or IgD, there are as a rule also present, and in general in a higher concentration, antigen-specific IgG antibodies in the patient's sample.
In the second place, the activity of rheumatoid factors (RF), that is to say antibodies against IgG which belong to various immunoglobulin classes, has to be suppressed because it can lead to falsification of the result. This falsification is possible because RF are bound to the antibody on the solid phase, and bound over the antigen-specific IgG antigen which is bound by the RF in turn, and thus a false-positive detection reaction is obtained.
It has been possible to eliminate both possibilities of interference by, for example, addition of anti-human IgG, gamma-chain (“RF adsorbent” of Behringwerke AG) to the sample (for example serum).
The invention relates to an immunochemical method for the determination of antibodies which are specific for an antigen and are of one of the immunoglobulin classes A, M, D or E in a fluid, with this fluid being contacted with a solid phase to which the antibodies against this immunoglobulin class, or a fragment of an antibody of this type, are bound, which results in the immunoglobulin of this class being bound to this solid phase, and this solid phase being contacted with the antigen, which carries a labeling means where appropriate, and with a labeled antibody or a labeled fragment of an antibody against the antigen if the antigen is unlabeled, and determination, from the amount of labeling means which is bound to the solid phase, of the amount of these antibodies which are specific for an antigen and are of one of the immunoglobulin classes, which comprises the solid phase being simultaneously in contact with the fluid containing the antibody which is to be determined and with the antigen, which is labeled where appropriate, there being addition of a substance which pr
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Nguyen Bao Thuy L.
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