Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1997-05-22
2001-02-06
Eyler, Yvonne (Department: 1642)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007100, C435S007900, C435S007920, C435S007940, C435S007930, C435S962000, C436S523000, C530S387300
Reexamination Certificate
active
06184042
ABSTRACT:
DESCRIPTION
The present invention concerns a method for the immunological determination of an analyte according to the sandwich assay principle and suitable binding substances for this. In particular the invention concerns an improvement of such sandwich methods in order to extend the measuring range and to reduce the Hook effect.
The quantitative determination of antigenic substances by means of immunoassays is known. A so-called sandwich assay is often used for this in which two antibodies directed towards the same or different epitopes of the analyte are incubated with a sample containing an analyte to be determined. In this method a first soluble antibody is directly or indirectly coupled to a signal-generating system i.e. a label whereas a second antibody (in a heterogeneous assay) is present coupled to a solid phase or is provided with a binding component such as e.g. biotin which is capable of binding to an appropriately coated solid phase.
The concentration of a number of diagnostically important proteins can vary within a wide range which means a wide measuring range is desirable or even essential for the analytics. Examples of such proteins are e.g. cancer parameters such as &agr; fetoprotein (AFP) and carcinoembryonic antigen (CEA) and also the pregnancy protein human chorionic gonadotropin (HCG). When determining such analytes it is diagnostically important on the one hand to obtain an exact value in high concentration ranges in order to be able to carry out a successful monitoring but on the other hand it must also be possible to carry out an exact determination in the lower concentration range for a qualitatively correct diagnosis (yes
o) which in turn can lead to fundamental therapeutic consequences.
A problem with high analyte concentrations in a sample to be examined is the so-called “Hook effect” which is understood as a decrease of the detectable signal at very high analyte concentrations. The reason for this is that normally in a heterogeneous sandwich assay format the soluble antibody and the solid phase antibody are present in an excess relative to the analyte to be determined so that the sandwich complexes can be formed and also detected essentially completely; however, in the case of a high analyte concentration a limited number of antibodies is faced by a very large number of analyte molecules. In the extreme case there is a deficit of solid phase antibody so that the analyte is only partially bound and moreover the fraction of analyte bound to the solid phase cannot be completely detected since labelled antibody is captured by the excess of analyte with formation of soluble detection antibodies: analyte complexes. This results in a reduction of the measured signal which can lead to a false negative test result.
One solution to the problem is of course to adequately dilute the sample to be examined. However, since in practice it is not known from the start when and to what extent a dilution has to be carried out, this means that several measurements with different concentrations have to be carried out which is undesirable for reasons of costs and because of the increased amount of work.
A further solution to this problem is to increase the concentration of both antibodies used in the sandwich assay. This however, results in a number of disadvantages such as for example additional possibilities of interference, a high blank value and an unfavourable shape of the calibration curve. Moreover the costs of each individual determination are also increased so that this possibility can also not be regarded as satisfactory.
Yet a further solution is disclosed in U.S. Pat. No. 4,743,542. This patent teaches that the calibration curve can be linearized at high analyte concentrations by addition of unlabelled first or second antibody or of mixtures thereof. This lowers the sensitivity in the lower measuring range but increases it at higher analyte concentrations resulting in an overall linearization of the calibration curve.
However, a disadvantage of the method according to U.S. Pat. No. 4,743,542 is the reduction of the sensitivity in the lower measuring range. Such a reduction is particularly critical when an accurate test result is essential in the lower measuring range for example in the HCG test in order to determine whether a pregnancy is present or not.
Hence the object of the invention was to provide a method for the immunological determination of an analyte in which the disadvantages of the state of the art are at least partially eliminated and which in particular enables a quantitatively exact determination over a wide measuring range while at the same time obtaining good results in the lower concentration range.
This object is achieved according to the invention by a method for the immunological determination of an analyte in a sample liquid according to the principle of a sandwich assay in which the sample liquid is incubated in the presence of a solid phase with at least two receptors capable of binding to the analyte to be determined in which the first receptor is soluble and the second receptor (a) is bound to a solid phase or (b) is capable of binding to a solid phase and the analyte is detected by determining the label in the solid phase or/and in the liquid phase which is characterized in that the first receptor is an oligomer of a binding molecule selected from antibodies, antibody fragments and mixtures thereof.
It has surprisingly turned out that the use of a soluble oligomeric antibody enables the Hook effect to be reduced and hence an extension of the measuring range. In general the sensitivity in the lower measuring range is retained or even improved. The lower limit of detection LLD according to Kayser (Fresenius “Zeitschrift für analytische Chemie”, volume 209, number 1, page 1-18, 1965) is used in the present description as a measure of the sensitivity in the lower measuring range.
The first receptor used according to the invention is an oligomer of a binding molecule selected from antibodies or/and antibody fragments and for the sake of simplicity is referred to as “oligomeric antibody” in the following. The prefix “first” or “second” antibody only serves the purpose of distinction in this description and does not for instance refer to an order of addition etc. The term “antibody” in the present invention is understood as an antibody with a single specificity such as a monoclonal antibody as well as a mixture of antibodies which are directed towards different epitopes of the same antigen such as a polyclonal antiserum. “Antibody fragment” is understood as any molecule which is derived from a complete antibody while retaining at least one paratope and which can for example be obtained by enzymatic or chemical treatment of an antibody or by genetic engineering. In particular an F(ab′)
2
fragment is understood as an antibody fragment.
The degree of oligomerization relative to a complete antibody or to an F(ab′)
2
fragment is at least two i.e. the minimum number of paratopes of an oligomeric antibody according to the invention is four. The minimum degree of oligomerization is preferably two to three. The maximum degree of oligomerization is up to 15, preferably up to 10 and more preferably up to 8. The degree of oligomerization is most preferably 2 to 8 and especially preferably 4 to 6.
The oligomeric first receptor is preferably used in a labelled form in the method according to the invention. This means that it is coupled directly or indirectly to a labelling group. In this connection a direct coupling is understood as a covalent incorporation of a detectable substance or of a molecule which on reaction with a suitable substrate generates a detectable substance. An example of the latter would be for example an enzyme which is covalently bound to the receptor optionally via a spacer. An indirect coupling denotes a configuration in which the first receptor according to the present invention and a detectable substance or a molecule that generates such a substance are capable of binding to one another via a specific binding p
Franken Norbert
Lenz Helmut
Neumann Ulrich
Boehringer Mannheim GmbH
Eyler Yvonne
Fulbright & Jaworski LLP
Nichols Jennifer
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