Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1991-10-04
2001-04-03
Eyler, Yvonne (Department: 1642)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S533000, C436S534000, C436S539000, C436S824000, C435S007100
Reexamination Certificate
active
06210975
ABSTRACT:
The present invention is concerned with an immunological precipitation process for the determination of a bindable analyte by incubation of a sample solution, which contains the analyte, with a specific receptor bindable with the analyte, as well as a reagent suitable therefor.
Immunological precipitation processes have long been used for the determination of bindable analytes. Use is made of the fact that many immunological reactions lead to formation of molecule aggregates or, in the case of an agglutination reaction, to particle aggregates, which differ considerably in the manner in which these diffuse light as compared to the starting materials. The concentration of these particles can be determined via of this property. For the quantitative determination of analytes, light scattering of particles present in a homogeneous medium, is used to measure the diffused light intensity (nephelometry) and to measure the loss of intensity of a light beam passing through the medium (turbidimetry).
A fundamental problem of all quantitative immunological precipitation processes results from the shape of the reaction curve. If antibody concentration is kept constant and increased amounts of antigen are added, then a typical curve results for precipitation (Heidelberger curve) (see
FIG. 1
of the accompanying drawings). In the zone of antibody excess, the concentration of the precipitate and thus the measurement signal increases. If further antibody is added the curve passes through a maximum and again decreases in the zone of the antigen excess. Based on this a measurement signal can be associated to two antigen concentration values. If the antibody is present in excess (C
2
), the measurement signal lies within the measurement range (C
0-C
M
) for which the ascending limb of the Heidelberger curve is suitable, then the false antigen concentration C
1
is read off. In the case of immunological methods of determination, this effect is also referred to as the “high dose Hook effect”, hereinafter briefly referred to as the “Hook effect”. For many analytes and especially proteins, the naturally occurring maximum physiological concentration lies far beyond the maximum of the Heidelberger curve so that these possibilities of error occur very frequently. In order to avoid these errors when determining the analyte it must be ascertained whether the measurement signal is present in the ascending or descending limb of the Heidelberger curve.
The oldest and most certain method was described by H. E. Schulze and G. Schwick, Prot. Biol. Fluids, 5, 15-25/1958, which provides for a double determination with two different sample dilutions. In the case of antigen excess, with the more highly diluted sample, a greater measurement signal is obtained than with the more concentrated sample.
An improved embodiment of this process provides for further addition of antibodies. When an excess of antigen is present a signal increase occurs (T. O. Tiffany et al., Clin. Chem. 20, 1005-1061/1974). By additional pipetting of antigen material of known concentration, antigen excess can also be recognized (J. C. Sternberg, Clin. Chem. 23, 1456-1464/1977).
Furthermore, several processes have been described which seek to recognize the hook effect by laborious computer-controlled evaluations. By determination of the period of reaction up to the appearance of the maximum reaction velocity for nephelometric measurements, it is possible to discriminate between antigen and antibody excess (DE-A-27 24 722; EP-B-0,148,463).
These processes all suffer from the disadvantage that either an additional pipetting step or computer-controlled evaluation is necessary and, in the case of automation, this automatically leads to an increase of the cost of the apparatus. A better alternative for avoiding erroneous interpretations in immunological precipitation processes would be to change the form of the Heidelberger curve so that, after the maximum, a plateau is reached and the appearance of the hook effect is completely avoided or is displaced into an antigen concentration range which no longer occurs under physiological conditions.
In U.S. Pat. No. 4,595,661 sandwich and nephelometric immunoassays are described which use highly specific antibodies and reagents normally present in an immunoassay, and also contain at least one further low-affinity antibody against the analyte in order to reduce the hook effect. The disadvantage of this process is obvious since, for each analyte to be detected, two specific antibodies with different affinities for the analyte must be prepared.
Therefore, it is an object of the present invention to provide a simple immunological precipitation process for the determination of a bindable analyte which avoids the hook effect.
Thus, according to the present invention, there is provided an immunological precipitation process for the determination of a bindable analyte by incubation of a sample solution which contains the analyte with a specific receptor bindable with the analyte, wherein to the test solution is added a non-ionic polymer from the group consisting of dextran having a molecular weight of at least 200,000, polyvinylpyrrolidone having a molecular weight of at least 100,000 and polyethylene glycol with a molecular weight of at least 10,000.
The present invention also provides a reagent for carrying out an immunological precipitation process for the determination of a bindable analyte in a sample solution, wherein, in addition to the materials necessary for the immunological precipitation process, it contains a non-ionic polymer from the group comprising dextran with a molecular weight of at least 200,000, polyvinylpyrrolidone with a molecular weight of at least 100,000 and polyethylene glycol with a molecular weight of at least 10,000.
The non-ionic polymer concentration in the test batch, i.e. the mixture of the sample solution, the specific receptor and the non-ionic polymer, is at least 1% by weight. Below this concentration, the action according to the present invention is no longer achieved. The upper amount is given by the appearance of non-specific turbidities resulting from high polymer concentrations. In the test batch, a non-ionic polymer concentration of 2 to 6% by weight has proved to be advantageous, a concentration of from 3 to 4% by weight being especially preferred. The reagent according to the present invention can be present as powder, lyophilisate or solution.
As non-ionic polymers, there e.g. polyethylene glycol, polyvinyl pyrrolidone or dextran can be used. Non-ionic polymers are obtainable in differing degrees of polymerisation, i.e. with different molecular weights. For the present invention, high molecular weight non-ionic polymers are preferred, the upper limit of the molecular weight depending upon the molecular weight at which the polymer is no longer sufficiently soluble in the test batch in order to be effective according to the present invention. For use in the process according to the present invention, polyethylene glycol has a molecular weight of at least 10,000, preferably a molecular weight of from 10,000 and 300,000 and most preferably a molecular weight of 40,000. Polyvinylpyrrolidone has a molecular weight of at least 100,000 and preferably of 360,000 to 750,000. Dextran is preferably used which has molecular weight of at least 200,000. Especially preferred is dextran with a molecular weight of from 500,000 to 1,000,000.
It was known to add polymers, such as polyethylene glycol, dextran or hyaluronic acid to immunological precipitation processes in order to increase the sensitivity and to accelerate reactions. Polyethylene glycol with a molecular weight of about 6000 is usually employed in a concentration of approximately 4% by weight in the case of nephelometric or turbidimetric determinations of an immunological precipitation reaction for these purposes (EP-B-0,148,463).
It was surprising that the addition of non-ionic polymers with the said molecular weights and concentrations avoids the hook effect as a disturbance which occurs in the case of high antigen conce
Karl Johann
Lang Fridl
Eyler Yvonne
Fulbright & Jaworski
Nichols Jennifer
Roche Diagnostics GmbH
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