Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-04-26
2001-04-17
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S007940, C435S188000, C435S810000, C435S962000, C435S970000, C436S518000, C436S524000, C436S528000, C436S162000, C436S169000, C436S807000, C422S051000, C422S067000
Reexamination Certificate
active
06218134
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a specific binding assay process for simple and quick qualitative or quantitative analysis of substances to be assayed in test samples and to a specific binding assay device suitable for the practice of the process.
More particularly, the present invention relates to an assay process and a device suitable for the practice of the process in which
(1) distribution of a label (signal substance generator) correlative to a detection means can be changed in response to the concentration of a substance to be assayed, in a matrix where a specific binding substance which binds specifically to the substance to be assayed is immobilized or included, through at least one specific binding reaction of the substance to be assayed with the specific binding substance, using a specific binding reaction-related substance as the label (signal substance generator) that binds to the specific binding substance in competition with the substance to be assayed or binds to the substance to be assayed directly and specifically without binding to the specific binding substance and can generate signal substances directly or indirectly and
(2) signals observed at the detection means can be modulated in response to the distance between the label and the detection means (that is, diffusion length of a signal substance), making use of a signal substance which is released from the label (signal substance generator) and directly or indirectly generates signals that are detectable only at the detection means.
BACKGROUND OF THE INVENTION
There are a number of known specific binding assay methods such as an immunoassay in which an antigen-antibody reaction is employed, a receptor assay in which a receptor is used, and a nucleic acid probe assay in which hybridization of complementary nucleic acid sequences is employed. Because of the high specificity, these assay methods are used frequently in various fields including clinical inspection and the like.
In general, these methods are divided into a heterogeneous method which requires a step for the isolation of unreacted substances after specific binding reactions and a homogeneous method that does not require such an isolation step.
The heterogeneous method is useful for the measurement of samples with relatively high sensitivity and can be regarded as a general purpose method, because it can detect degree of the specific binding reaction without receiving interference of unreacted substances. This method, however, requires complex handling for the removal of unreacted substances and, in some cases, requires special tools or instruments such as a washing apparatus. Because of this, it is still necessary to improve this method in terms of its simplicity and rapidly.
With the aim of improving the handling simplicity of the homogeneous type assay method, various techniques have been developed, which include agglutination method, EMIT method, proximal linkage immunoassay such as an enzyme channeling technique, immunochromatographic assay and the like. These techniques, however, are still inferior to the heterogeneous method in terms of efficiency and general purpose use.
For example, the agglutination type methods which are effected by the formation of aggregates by antigen-antibody reaction or by the inhibition of the aggregate formation, such as a gel diffusion technique, an erythrocyte agglutination technique, a latex agglutination technique and the like, have considerably inferior sensitivity and accuracy in comparison with other methods in which labels such as enzymes are used.
Examples of homogeneous type methods which are effected by the use of labels such as enzymes include: EMIT method and the like which are effected by the modulation of enzyme activity, itself based on a specific binding reaction; and proximal linkage immunoassay in which signal modulation occurs when two types of labels having mutual relation get close due to a specific binding reaction. In the case of the EMIT method and related techniques, it is necessary to modify an enzyme, a coenzyme component, an enzyme inhibitor or the like with a substance to be assayed (or an analogue of the substance to be assayed) while the activity of the enzyme or the like is kept constant and to induce a significant modulation of the enzyme activity through a binding reaction of the modified product with a specific binding substance. Because of such requirements, this method is inferior to other methods in terms of sensitivity and general purpose use and, in some cases, cannot be used depending on the physical properties (molecular weight, configuration, solubility and the like) and chemical properties (reactivity, position of functional groups and the like) of the substance to be assayed. On the contrary, the proximal linkage immunoassay can sometimes be effected by certain labeling techniques which are generally used in the heterogeneous method and therefore is superior to the EMIT method and the like in terms of general purpose use. In addition, from the viewpoint of specific binding reaction and detection of signals, increase in the sensitivity and decrease in the measuring time can be expected as the concentration of specific binding substances including labeling substances increases. Such a concentration increment, however, increases approaching frequency of labels independent of the specific binding reaction. Such properties which are contrary to each other become a significant factor that limits the sensitivity. An example of the proximal linkage immunoassay is the enzyme channeling technique in which two types of enzymes are used that catalyze two continued reactions such as formation of a product by a first enzyme and utilization of the product by a second enzyme as its substrate. Processes in relation to such a technique have been disclosed for instance in GB 2013986, EP 32286, EP 75379
, Analytical Biochemistry
(vol.106, pp.223-229, 1980) and the like. However, each of these prior art processes is based on the modulation of the total enzyme reaction rate, which is effected by a difference between a time when a labeled enzyme exists in a free state and a time when a microscopic environment is formed by a proximity effect of labels by a specific binding reaction so that channeling of the labeled enzymes can be carried out, or when the labeled enzyme undergoes specific binding to a dispersion type discrete particulate carrier (dextran particles or the like) or to a non-dispersion type carrier (filter paper or the like) which provides a channeling environment. In consequence, a free labeled enzyme in the liquid phase could approach the solid phase by free diffusion, and its non-specific reaction would cause a problem when the concentration of the labeled enzyme is high. Another problem of this technique is that, since a first enzyme can continue its reaction independent of the degree of proximity of a second enzyme, the reaction product of the first enzyme accumulates inside the measuring system when an environment for its channeling with the second enzyme is not formed. Such an accumulation of the product formed by the reaction of the first enzyme also causes acceleration of non-specific reactions. In order to solve such problems, a scavenger is used which can remove excess amounts of the first reaction product. However, addition of a scavenger in an effective amount causes a certain competitive inhibition effect on the whole reaction. Application of such a scavenger, therefore, cannot be regarded as a general purpose countermeasure.
A different type of assay method has been reported which includes a step in which a test sample presumably containing a substance to be assayed is permeated and developed, together with or separately from a labeled specific binding substance, in a chromatographic area that comprises a porous or particulate-packed type carrier to which a specific binding substance is immobilized. Such a method, or an immunochromatographic assay, is advantageous in some cases from the viewpoint of sensitivity and measuring time because
Kanamori Toshinori
Nobuhara Masahiro
Yamauchi Tadakazu
Birch & Stewart Kolasch & Birch, LLP
Chin Christopher L.
Do Pensee T.
Mochida Pharmaceutical Co. Ltd.
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