Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is particulate and the particles are of...
Patent
1990-08-03
1993-06-15
Kepplinger, Esther L.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is particulate and the particles are of...
435973, 436524, 436528, 436531, 436534, 436536, 436538, 436800, 436805, G01N 33538, G01N 33546
Patent
active
052197638
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for accurately and specifically determining in a liquid or semi-liquid, at least one biological, organic or drug soluble substance, even if it is present in extremely small amounts by means of a suitable agglutination reaction.
It is known to detect and to determine soluble substances and particularly peptides or proteins by an agglutination reaction or an agglutination inhibition reaction.
The agglutination reaction is the direct consequence of the fixation of an agglutinating antibody on a cell, for example, and thus calls for antigen-antibody complexes or hormone receptor complexes having agglutinating properties.
Nonetheless, it should be recalled that although these reactions may be easy to produce, their interpretation by the naked eye is mostly delicate and requires the presence of a large amount of substance to be assayed in the specimen, for interpretation to be easy.
An agglutination reaction can be contemplated in various biological contexts; in particular in the field of immunology, agglutination is the manifestation of the formation of a complex.
There exist so-called direct methods (antigen/antibody complex), so-called indirect methods or methods of inhibition by competition; there are also known assay methods of the so-called "sandwich" or bisite type; in these "sandwich" type methods, two antibodies are used, one to capture the antigen, the other to reveal its presence, if this antigen is in fact present and has been captured by the first antibody. Generally, the first antibody is fixed to a solid support (plate, bead . . . ), the second antibody being conjugated with a reaction "developer" such as fluorochrome, radio-isotope, or enzyme.
In an agglutination reaction, red blood cells coated with an antigen serve for detecting corresponding specific antibodies (hemagglutination). An extension of the agglutination methods has consisted of using plastic beads: in the case of a positive reaction, the appearance of a macroscopic "granulation" (passive agglutination) is observed. A reverse procedure has also been described: the antibodies are fixed to beads and serve to detect the corresponding antigens. The sensitivity of such a method is high, but, as has already been mentioned above, the interpretation of the result is not objective and is variable as a function of the observer, the amount of agglutination being a function of the amount of antigen present for a certain range of concentrations of this antigen.
In order to overcome this drawback of agglutination methods and in order to make them quantitative, a certain number of authors have proposed the counting of the non-agglutinated particles by means of a suitable particle counter especially a blood cell counter.
MASSON et al (Particle Counting Immunoassay, Meth. Enzymol., 1981, 74, 106-139) propose a method of determining various antigens (proteins, peptides . . . ), antibodies, haptens and immunocomplexes by means of an agglutination reaction between latex particles 0.8 .mu.m in diameter coated with antibodies or suitable antigens and said antigens or antibodies or haptens or immunocomplexes, and the counting of the non-agglutinated particles by means of a particle counter, particularly a red blood cell counter which is a small angle defracted light detector. The counting of the non-agglutinated particles enables evaluation of the extent of the agglutination reaction and, consequently, the amount of product to be determined.
The detector is parametered so as to take no account electronically of particles whose diameter is less than 0.6 .mu.m and particles whose diameter is greater than 1.2 .mu.m (agglutinated particles).
MASSON described various elements of this method in a certain number of patents:
U.S. Pat. No. 4,062,935 (1977), claims a method of detecting antigen/antibody complexes which comprises the addition to the sample of a rheumatoid factor (RF) and material which agglutinates RF on contact, then detection of the amount of agglutination of said material compared with a standard mixture
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Chemunex
Chin Christopher L.
Kepplinger Esther L.
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