1. Field of the Invention
The invention concerns a multivalent dextran reagent for use in a precipitation test for the determination of a specifically bindable substance comprising dextran to which several molecules of a receptor R.sub.1 which is capable of specific binding to the substance to be determined or of the specifically bindable substance or of an analogue of this substance are bound or can be bound.
The invention also concerns a precipitation test for the determination of a specifically bindable substance in which the multivalent dextran reagent is used in addition to reagent which are usually necessary for a precipitation reaction such as buffer, substance for reducing interference, reaction accelerator or detergent.
2. Description of the Prior Art
Very many substances occur in body fluids and tissues which are capable of binding to a specific binding partner and which serve as parameters for particular diseases or for the state of health of the body. These include haptens such as hormones, proteins such as C-reactive protein (CRP), glycated proteins and viral proteins as well as antibodies. When monitoring drug treatment it is often necessary to determine pharmaceutical substances in blood. The bindable substances occur in a wide variety of concentrations in the body fluids or tissues. Various proteins such as IgG, IgA or apolipoproteins occur in high concentrations while for example hormones or drugs or even other proteins may be present in very low concentrations. The detection of these substances can be carried out by a precipitation test. Specifically bindable substances which are at least bivalent, i.e. have at least two epitopes which bind to the receptor in the precipitation test, can be precipitated directly by the addition of a receptor capable of specific binding. In order to increase the sensitivity of the nephelometric or turbidimetric measurement it is often necessary to bind the receptors capable of specific binding to highly polymerized particles e.g. latex or red blood corpuscles. Such detection methods with a turbidimetric evaluation are denoted agglutination tests and have been described in Eur. J. Biochem. 30 (1971), 553-560.
Low-molecular substances, such as e.g. haptens, which only have one epitope to which the specific receptor binds in the precipitation or agglutination test cannot be detected with the direct test procedure for the precipitation or agglutination test since no crosslinking can form which leads to precipitation. An immuno-precipitation test is known from EP-A-0 079 962 which utilizes a competitive test procedure in which the solution containing the haptens to be determined is brought into contact with an albumin coated with hapten. Addition of antibodies capable of binding to the haptens leads to a precipitation reaction. Since the hapten bound to the albumin competes with the hapten present in the sample, the precipitation reaction is smaller the more hapten is present in the sample. Other proteins are described as hapten carriers in the state of the art e.g. IgG, latex particles or synthetic polymers such as e.g. polyaspartate (EP-A-0 317 796).
All these agglutination tests or precipitation tests still have some considerable disadvantages. Pure precipitation tests have a sensitivity which is too low for some parameters. Therefore specific receptors are bound to latex particles in order to increase the sensitivity of agglutination tests. The coupling can, on the one hand, lead to an impairment of the reactivity of the receptor itself and, on the other hand, the addition of reaction accelerators such as e.g. polyethylene glycol, which has to be added to avoid a long incubation period, can lead to spontaneous agglutination reactions of the latex particles. It is also difficult to exactly control the number of receptors coupled to the individual latex particles. As a consequence considerable variations can occur between the individual lots. Binding of unspecific factors from the sample to the surface of the latex particles can lead to inaccuracies in the measurement.
Difficulties can also occur when using carrier-bound haptens such as hapten-albumin or hapten-IgG. Possible interfering factors are anti-IgG and anti-albumin antibodies in the serum. Also in this case it is difficult to exactly control the number of haptens on the individual molecules in particular when using natural molecules such as proteins. Moreover these materials are not stable on storage to an extent which would allow their unlimited use. These materials are not optimal with regard to their solubility properties and their tendency to denature and cannot therefore be used universally.
Up to now it has been necessary depending on the test procedure, whereby one has to differentiate between a direct or competitive test procedure, on the substance to be detected such as haptens, antigens or antibodies and on the concentration of the substance to be detected to test which of the possible materials available is the optimal carrier material in each case for binding the specific receptor or the specifically bindable substance for an agglutination test or a precipitation test with carrier-bound haptens. A carrier material which can be used universally for all test variants, all substances to be detected and concentrations and which in addition has a long shelf-life, good solubility properties and a low tendency to denature, and which also allows the use of different molecular sizes and a wide range of incorporation rates is not known up to now.
USPTO Backgrounds
USPTO Backgrounds