Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody – Through a gel
Patent
1991-10-09
1995-07-04
Woodward, Michael P.
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
Through a gel
436514, 436518, 436525, 436534, 436823, 422 57, G01N 33559, G01N 33543, G01N 33553, G01N 2182
Patent
active
054299519
DESCRIPTION:
BRIEF SUMMARY
This invention is concerned with improvements in and relating to radial immunodiffusion and like techniques.
Several methods of measuring the concentration of a proteinaceous antigen in a sample are already known. A first of these methods is well-known and widely used and is normally referred to as immunodiffusion. In that method a sample of the material at a predetermined dilution is introduced into a well in a plate of agarose gel containing an antibody specific to that antigen, the antibody being present at a predetermined concentration in the gel. The sample diffuses radially from the well into the gel, where the antigen becomes releasably bound to the antibody. This results in the formation of a matrix which is visible as a ring, or sometimes as a disc, concentric with the well. With the passage of time the diameter of the ring or disc increases until a maximum diameter is reached, and it is usual for the inner part of the ring or disc to become less distinct or to disappear as the maximum outer diameter is reached. Experiment has shown that the concentration of antigen in the original sample is directly proportional to the maximum diameter of the ring or disc. The method is described in the following paper: Mancini G., Carbonara A. O. and Heremans J. F., Immunochemistry, 2, 235 (1965).
In a converse of that first method, which is of use in assessing the concentration of antibody in a sample, the agarose gel contains a predetermined concentration of the antigen, while the sample contains an unknown concentration of the complementary antibody.
The visible matrix is most clearly formed when the concentrations of the protein and the antibody are optimal. At those optimal concentrations most of the antibodies become bound to two separate molecules of antigen while most of the antigen molecules become bound to two or more separate antibodies so that extended aggregations of molecules are formed, those aggregations constituting the visible basis of the matrix. Where there is an excess of antibodies few or none of the antibodies are able to link together separate antigen molecules. Likewise where there is an excess of antigen molecules, some pairs of antigen molecules may each be linked together by an antibody molecule but those linked pairs are not linked to form large visible complexes as insufficient antibody molecules are present.
For a matrix to interfere with the direct passage of light through a gel, it must comprise individual aggregations of molecules each of which is of an extent which is at least about one twentieth of the wavelength of the light. In practice, noticeable scattering occurs when there are aggregations each of which is some 200 to 400 nm in diameter. As an individual antibody molecule or a typical individual antigen molecule is only a few nm across, e.g. about 5 to 8 nm across, it means that no scattering of light that is visible to the eye occurs until there are formed aggregations of molecules each consisting of a very large number of molecules, typically at least a few million molecules. It also means that the concentration of antibody and antigen molecules in the interstices of the gel is relatively high. This in turn presupposes an adequate concentration of the antibody (or conversely of the antigen) in the gel initially.
While that type of method of radial immunodiffusion is extremely valuable for some purposes it does nevertheless suffer from the drawback that its sensitivity is limited owing to the need to enable relatively large aggregations of molecules to be formed. Moreover, the method is slow at low concentrations.
A second known method of measuring the concentration of proteinaceous antigen in a sample makes use of a liquid reagent comprising a suspension of latex particles coated with antibodies. The particles are sufficiently small that their presence does not greatly reduce the clarity of the reagent. In use, however, when a proteinaceous antigen is added to the reagent, the antigen links some of the coated particles together so that aggregations are formed which not
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Dunbar, B., pp. 143-150, From "Two-dimensional electrophoresis & immunol. techq." Plenum Press N.Y. 1987.
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