Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-06
2002-12-03
Chin, Christopher L. (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
06489131
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119 of German Application Ser. No. 198 20 239.3 filed May 6, 1998 and of German Application Serial No. 199 13 117.1 filed Mar. 23, 1999.
DESCRIPTION
The invention concerns a method for the determination of an analyte in which rheumatoid factors of rheumatoid-factor-like substances are added as an interference reducing reagent to reduce or avoid a hook effect. The invention in addition concerns suitable reagent kits for carrying out the method.
So-called sandwich assays in which two receptors directed against identical or different epitopes of the analyte are incubated with a sample containing the analyte to be determined, are frequently used for the quantitative determination of analytes in a sample. In this method a first soluble receptor is preferably directly or indirectly coupled with a signal-generating system i.e. with a label, whereas—in a heterogeneous detection method—a second receptor is present coupled to a solid phase or is provided with a binding component such as e.g. biotin which is able to bind to a suitably coated solid phase.
The analyte concentration in the sample can vary considerably for a number of diagnostically important parameters which means that a broader measuring range is desirable or even necessary. When such analytes are determined it is, on the one hand, diagnostically important to obtain a value in high concentration ranges which is as accurate as possible. On the other hand, it must also be possible to carry out an exact determination in low concentration ranges to enable a qualitatively correct yes
o diagnosis which in turn can have fundamental therapeutic consequences.
A problem with high analyte concentrations in a sample to be examined is the so-called “high dose hook effect” which is understood as a decrease of the measured signal at very high analyte concentrations. In this case a particular measured signal can be caused by two different analyte concentrations. This “hook effect” considerably limits the application of sandwich assays.
The occurrence of the hook effect is particularly serious in one-step sandwich assays. All tests are referred to as a one-step assay in which the analyte to be determined is reacted with at least two receptors that are specific for it i.e. form a sandwich with it in the same solution and in this process it is for example detected by immobilization on a solid phase in contrast to a sequential reaction procedure in which, after reaction of the analyte with a first specific receptor and immobilization of the complex that is formed on a solid phase, the non-bound analyte is removed by washing the solid phase before the reaction with a second specific labelled receptor.
Sandwich assays can also be carried out to detect antibodies, for example as bridge tests, in which an immobilized or immobilizable antigen that is specific for the antibody to be detected and a second labelled antigen are used. Such bridge test or double-antigen sandwich determinations are described for example in EP-A-0 280 211.
Analyte determinations utilizing the DSAP (double antibody solid phase) sandwich principle in which the two analyte-specific receptors are soluble and the complex with the analyte is immobilized on a solid phase using an additional receptor, are also one-step tests in the above sense and exhibit a hook effect.
Numerous attempts have been made to detect or avoid the hook effect e.g. by carrying out the assay in a two-step or multiple step method in which a wash is carried out after the first reaction step (Hoffmann et al., Clin. Chem. 30 (1984), 1499). However, a disadvantage of this method is that it involves more work, the total incubation time is increased end furthermore there is often a loss of sensitivity and it may not be possible to completely reduce the hook effect.
Another proposal was to carry out several determinations on a single sample with different dilutions in each case. However, this method also increases the amount of time and work that is needed and leads to increased costs for the user.
Hoffmann et al. (1984) supra additionally describe a method of determination for a one-step sandwich test in which the reaction of the analyte with the receptor is monitored kinetically and the values are stored in a computer. This enables measurement results which are caused by the hook effect to be distinguished from other values. A disadvantage of this method is that it requires extremely complicated and expansive computer analyses in order to recognize whether a hook effect is present in the determination.
Other proposals which have been made to reduce or avoid the hook effect also result in considerable disadvantages. Thus a reduction of the sample volume generally leads to a considerable loss of sensitivity. An increase of the receptor concentration (solid phase binding receptor or/and labelled receptor) often leads to a considerable increase in costs and, if the concentration of the labelled receptor is increased, to blank value problems and thus to a loss of sensitivity. Furthermore an increase of the receptor concentration only has a relatively low potential for reducing the hook effect.
Furthermore the addition of unlabelled antibodies or antigens to the test reagent was for example proposed in U.S. Pat. No. 4,595,661, U.S. Pat. No. 4,743,542 or EP-A-0 617 285. However, this also usually leads to a loss of sensitivity and to a considerable increase in the reagent costs.
EP-A-0 572 845 discloses a method for the immunochemical determination of an analyte in a sample by means of a first specific binding partner in which the specific binding partner is immobilized on a carrier and the extent of binding of the analyte to the specific binding partner is determined using an additional specific binding partner which directly or indirectly carries a label and in which a binding factor is additionally added in the method which has more than one site that is capable of binding to the analyte, has no affinity for the immobilized specific binding partner and is not labelled. Examples of such binding factors are monoclonal and polyclonal antibodies as well as fragments thereof, lectins or conjugates of several lectins or conjugates of lectins with analyte-specific antibodies or fragments thereof. When the analyte is an antibody, the antibodies used as binding factors are not homologous to the analyte antibodies and are directed against the specific immunoglobulin class of the analyte antibody. The use of rheumatoid factors to determine human antibodies is neither disclosed nor made obvious.
According to EP-A-0 572 845 the formation of an immune complex is independent of whether the binding faster is present or not. Furthermore it is emphasised that, due to the back reaction known to a person skilled in the art, the complex of analyte and specific receptor can disintegrate again and thus be unavailable for the detection reaction which severely limits the sensitivity. For this reason the diagnostic performance of the test is limited if the back reaction rate between the immobilized receptor and the analyte to be detected is very high. Such a back reaction rate is of particular importance in the lower measuring range i.e. at low concentrations of the analyte or in the case of low affinity anti-analyte antibodies.
Hence the object of the present invention was to at least largely avoid the above-mentioned disadvantages of the prior art. This object is achieved by adding polyclonal or/and monoclonal rheumatoid factors or rheumatoid-factor-like substances to the test mixture as interference reducing reagents. In this manner it is possible to reduce or completely avoid false measurements caused by the hook effect without involving additional time and work and without loss of sensitivity.
Hence a subject matter of the present invention is a method for the determination of an analyte in a sample which is characterized in that rheumatoid factors or rheumatoid factor-like substances are added to reduce interference. The determination is pref
Donie Frederic
Ofenloch-Hähnle Beatus
Wehner Rainer
Amick Marilyn L.
Chin Christopher L.
Cook Lisa V
Roche Diagnostics GmbH
Woodburn Jill L.
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