Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is a biological cell or cell fragment
Reexamination Certificate
1999-09-20
2001-10-16
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is a biological cell or cell fragment
C435S005000, C435S007100, C435S007250, C436S512000, C436S513000, C436S518000, C436S521000, C436S548000, C436S016000, C436S809000, C436S810000, C436S828000
Reexamination Certificate
active
06303390
ABSTRACT:
FIELD OF THE INVENTION
The invention lies in the field of bloodgroup serology and relates more particularly to a method and a test kit for determining bloodgroup antigens, or antibodies directed thereto, in a sample.
BACKGROUND OF THE INVENTION
The purpose of bloodgroup assays and antibody examination in clinical work is often to obtain compatible erythrocyte preparations for transfusion. The invention concerns the detection and identification of antibodies and antigens and can be used for bloodgroup assay, antibody screening and identification, and performing cross tests. The test is based not on hemagglutination but on the solid-phase principle, without involving washing steps.
In a bloodgroup, specific antigenic determinants on the cell membrane are involved. The information for expressing bloodgroup antigens is fixed on a gene level and heritable. Bloodgroup antigens can give rise to the formation of antibodies. Specific antibodies to bloodgroup antigens are mostly formed after immunization with the corresponding antigen. An exception to this is formed by the so-called “naturally occurring” antibodies which without apparent immunization are demonstrable in the serum (anti-A and anti-B). The presence of antibodies plays an important role in blood transfusion, pregnancy and auto-immunity.
Antibodies can be classified in different ways:
1. xeno-antibodies, allo-antibodies and auto-antibodies;
2. regular and irregular antibodies;
3. naturally occurring antibodies;
4. complete (IgM) antibodies and incomplete (IgG) antibodies.
Blood transfusion reactions which are induced by allo-antibodies to erythrocytes are called hemolytic transfusion reactions because they are mostly accompanied by an often very strongly accelerated breakdown of erythrocytes. The point is therefore to prevent hemolytic transfusion reactions by careful bloodgroup examination.
Bloodgroup antibodies are nearly always immunoglobulins of the IgG type or the IgM type. The antigen-antibody interaction is dependent inter alia on ionic bonding, hydrogen bridges and hydrophobic effects (displacement of water). The strength of a binding between a binding site of an antibody and an epitope is designated as ‘affinity’. Antibodies which are able to agglutinate erythrocytes under all conditions are called agglutinins or complete antibodies (mostly IgM). Antibodies which do bind to erythrocytes but give no agglutination (sensitization) are called incomplete antibodies (mostly IgG).
The detection of erythrocyte antigens and corresponding antibodies often takes place by means of agglutination reactions. Agglutination reactions can be allowed to take place in a physiological salt solution. In practice, this is not always optimal. A number of tests can be rendered more sensitive by using a number of aids, such as the use of medium having a low ionic strength (low ionic medium), proteolytic enzymes (e.g. bromelin, papain or ficin), polycations (e.g. polybrene), macromolecules (e.g. albumin), or polymers (e.g. polyethylene glycol, PEG).
An important and widely applied test is the antiglobulin or Coombs test. The antiglobulin test is based on the principle that erythrocytes loaded with, for instance, antibodies of the IgG type can be agglutinated by antiglobulin serum. This is the most important test for demonstrating incomplete antibodies. The antiglobulin test was described by Moreschi in 1908 (Zbl. Bakt 46: 49) and reintroduced in 1945 by Coombs et al. (Lancet 2: 15, Brit. J. Exp. Path. 26: 255). The test can be distinguished into three phases. The first phase is the sensitization phase. During this phase, antibodies bind to the corresponding antigen structures on the erythrocytes (sensitization of erythrocytes). When the binding is optimal, the second phase, viz. the washing phase, takes place. In this phase, all non-bound antibodies are removed from the incubation mixture. Insufficient removal of non-bound antibodies can lead to inactivation of the antiglobulin serum in that these antibodies bind to the antiglobulin. The third phase is the antiglobulin phase, in which antiglobulin is added to the washed sensitized cells, so that the sensitized cells are coupled to each other (agglutination of the erythrocytes).
There is a large variety of serological tests. The most important techniques at present are the tube method, the column test and tests in microtiter plates. A distinction can be made between techniques that are based on hemagglutination and techniques that are based on the solid-phase principle.
a. Tests based on agglutination:
The tube test is a widely used test which also allows prolonged incubations with antibodies. After the reaction with antibodies, the erythrocytes can be settled or be centrifuged to accelerate the agglutination reaction.
In performing the Coombs test, it is necessary, before adding the antiglobulin serum, to wash very thoroughly and frequently. The antigen-antibody reaction is evaluated by gently tapping the tube and then rotating the tube (tip and roll) so that any agglutinates that have formed are dislodged from the tube. The reading of the test must be done promptly by an experienced person and the result of the test cannot be preserved. Because the reading of the test occurs manually, its reproducibility is not optimal either. The disadvantage of this method is that it is difficult to automate.
As indicated, the washing step in the Coombs test takes up much time. Graham et al. (Transfusion 1982, 22: 408; P. L. Mollison, Blood transfusion in clinical medicine, Blackwell, Oxford, 1983, p. 512) developed a new principle which rendered the washing step redundant. This principle was used by Ortho Diagnostic Systems Inc. in a test system (Simwash). According to this system, erythrocytes are separated from the serum by means of a centrifugation step. The separation is based on the fact that the specific gravity of serum (1.03) is lower than that of erythrocytes (1.09). Now, if a mixture of cells and serum is applied on top of a layer of medium of a density between the density of the cells and that of the serum, the erythrocytes will be separated by a centrifugation step from the serum (containing the non-bound antibodies). Thus the erythrocytes are centrifuged from the original incubation mixture. In this way a triple or quadruple washing step is reduced to a single centrifugation step. Then the sensitized cells can be incubated with antiglobulin serum. However, the system proved not to be sensitive enough.
A further simplification of serological tests has been carried through by centrifuging the erythrocytes through a column of (Sephadex) gel or glass beads. The use of transparent, inert, solid particles for distinguishing agglutinates and non-agglutinates was already described by Dalton et al. in 1970 (Becton Dickinson & Co., U.S. Pat. No. 3,492,396). The system whereby gel material is used for the detection of erythrocyte-antibody reactions was described by LaPierre et al. (Transfusion 1990; 30: 109-113, European Patents 0 194 212 and 0 305 337). This system combines the principles of Simwash and the use of solid inert particles to discriminate between agglutinates and non-agglutinates. In this test system, use is made of small columns filled with Sephadex gel. Use can be made of columns not containing any antibodies (e.g. for reverse ABO typing). As a second possibility, a gel column can also contain antibodies which are directed to certain erythrocyte antigens (e.g. for typing). For the antiglobulin test, use is made of gel columns which contain antiglobulin serum. After an incubation the gel columns are centrifuged. In the case of a negative reaction, all erythrocytes will end up at the bottom of the tube; if the test is positive, the erythrocyte agglutinates will be caught on top of or in the gel. In the case of weak reactions, erythrocytes will sediment partly. A major advantage of this test is that in case of the antiglobulin test no washing steps are needed anymore. In fact, during the centrifugation a separation of erythrocytes and serum or plasma constituents takes place. A second adv
Den Boer Pieter Johannes
Van der Donk Eric Marinus Maria
Van Eijk Ronald Victor Wilhelmus
Do Pensee
Hoffman & Baron LLP
Le Long V.
Stichting Sanquin Bloedvoorziening
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