Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1984-09-28
1987-11-24
Nucker, Christine M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
422 69, 435174, 435177, 435179, 435180, 435181, 435182, 435805, 436528, 436529, 436530, 436531, 436535, 436541, 436807, G01N 3352, G01N 3353, G01N 33544, C12N 1104
Patent
active
047089326
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improved and simplified method of performing analytical determination methods based upon biospecific reactions in heterogeneous systems, as well as a matrix to be used therefor.
A great number of biochemical determination methods are based upon biospecific affinity reactions between a receptor and a ligand. As examples of antigen och antibody, enzymatic determination methods, such as enzyme-substrate or enzyme-co-enzyme, lectin-sugar, etc. Usually the ligand is the component to be determined and is dissolved in a liquid, while the receptor is immobilized on a carrier in contact with the liquid. By making a known quantity of a ligand labelled with a detectable group, or a so-called marker, compete with the non-labelled ligand or, alternatively, adding an excess of labelled ligand to the carrier after the reaction with the non-labelled ligand, the content of non-labelled ligand may be determined.
In, for example, radio-immunoassay or the so-called RIA-technique radioactive isotopes are used as markers. In this technique, for example, antigen molecules in a sample are caused to compete with added isotope-labelled antigen molecules for the available sites on antibodies bound to a carrier. After the reaction non-bound material is removed by washing, and the amount of remaining radioactivity on the carrier indicates the concentration of antigen in the sample. Alternatively, in accordance with the above, the antigen sample may be added to the carrier before the addition of the isotope-labelled antigen is added.
As carriers small spheres of gels or cellulose, small paper discs or simply the inside of a test-tube have been used. The actual biospecific reactions in question, however, proceed relatively rapidly, and in the heterogeneous reaction systems obtained with the above mentioned carrier types the transport phenomenons in the solution will therefore be determining for the total reaction rate and thereby the time it takes for the reaction to be completed to such an extent that a measurement may be made. In the free liquid volume there is a transport, i.e. an equalization of concentration differences, through convection, while at the interface there is always an adsorbed liquid layer through which the transport is effected only through diffusion. The thickness of this reaction rate determining diffusion layer may be reduced by agitation, and stirring or vibration of the reaction system is therefore used to reduce the diffusion dependence. Another method used to increase reaction rate is to enlarge the surface area of the carrier. Thus, e.g., a substantial independence of diffusion has been achieved without agitation by forming the carrier as micro-sized gel particles.
These methods for reducing the diffusion dependence are, however, associated with disadvantages. Thus, the use of agitation requires special apparatus and the reproductibility will not always be satisfactory due to the difficulty of maintaining constant agitation conditions. If, on the other hand, the material transfer rate is increased by surface enlargement of the carrier, as, e.g., for the above mentioned microgel particles, the separation and wash steps will instead be more complicated. In addition centrifugation is necessary to separate the liquid phase from the carrier.
The present invention relates to a new method of optimizing the rate of biospecific affinity reactions in a heterogeneous system, which method at the same time provides a more simplified and more reliable analysis procedure without the use of neither agitation nor centrifugation. The invention is based upon a combination of surface area enlargement and reduction of the thickness of tChe diffusion layer, namely upon the concept that all the reactions are carried out completely within an open-pored, liquid-absorbing matrix body, in the pores of which the receptor is immobilized and which has limited void dimensions selected such that the distance between the solid phase surfaces thereof confines the diffusion layer to such an extent that the r
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Axen Rolf E.
Bjorkman Rune
Kaj Goran L.
Nucker Christine M.
Pharmacia AB
Philpitt Fred
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