Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-01-11
1998-06-30
Huff, Sheela
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 72, 435 725, 435 721, 435 79, 435 792, 436518, 436519, 436520, 436521, 436522, 436827, 436828, 436820, 436 63, G01N 3353, G01N 33567, G01N 33555, G01N 33542
Patent
active
057732221
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to a solid phase immunological assay and in particular to a solid phase serological assay for the screening of blood samples.
Immunoassay, and in particular serological assay, is an important and routine aspect of clinical science. The ability to detect blood group specific antibodies in serum of patients prior to transfusion is particularly important. Prior to transfusion, donor and patient blood samples are phenotypically characterised for the major blood group antigens A, B, O, and Rh(D), followed by a further screen for atypical antibodies in the patients' serum such as those with Duffy, Kidd, Kell, MNS and other Rh specificities. Detection and subsequent identification of such irregular antibodies facilitates the selection of appropriate blood for transfusion, i.e. that which does not express antigens against which such irregular antibodies are directed. Transfusion with suitably selected blood avoids transfusion reactions in the patient, which range from a mild febrile reaction to death.
Known serological assays rely on red cell agglutination in liquid suspension for the detection of red cell antigen-antibody interactions. Such tests for antibody detection in patients' sera are essentially manual tests, and are highly subjective, requiring the operator to discriminate visually whether or not agglutination has occurred. Many antibodies which can give rise to clinically significant transfusion reactions if antigen positive blood is transfused in vivo are present at low concentrations and will only promote weak agglutination in vitro, even if undiluted serum/plasma is tested. The performance of such tests in practice shows them to be insensitive and inaccurate, as weak agglutination reactions can easily be missed by less skilled workers.
In other fields solid phase immunoassays are used in which antigen preparations are immobilised on a solid support and exposed to the test sample containing antibodies, whereupon bound antibody is subsequently detected for example by means of an enzyme or radio-labelled secondary antibody. This has the advantage that it is readily adapted to automation and by appropriate choice of a labelling agent, results can be quantitated. Such techniques are highly sensitive.
An application of solid phase techniques to antibody screening for irregular blood cell antibodies has been made using immobilised red cells which are reactive with the target antibody. Such techniques use antiglobulin bound to red cells as a means of detecting an antibody-antigen interaction, by a binding reaction between the antiglobulin and the immobilised target antibody. Such red cells are referred to as `indicators`. Red cells are prepared for use as indicators either by direct chemical coupling to antiglobulin using, for example, chromic chloride, or by means of an antibody to a red cell antigen, such as anti-Rh (D), which binds both to the red cells and to the antiglobulin reagent. In these systems, a positive reaction is indicated by adhesion of indicator red cells across the solid surface. When the reaction is carried out in a microplate, or other surface provided with recesses, the indicator cells may be centrifuged under appropriate conditions of g force and time onto the surface whereupon in a positive reaction, a monolayer of red cells forms around the well/recess, and in a negative reaction, a button of red cells is formed in the bottom of the well. Reaction is detected by means of visual examination of the indicator red cells, in which case a subjective interpretation of the red cell pattern is required. Automated objective reading of indicator red cell patterns requires image analysis equipment, and the patterns obtained vary with the centrifugation conditions used.
If, however, aqueous labelled, for example enzyme-labelled, secondary antibodies are used as the detection system for antibodies to red cell antigens instead of the indicator red cell labelled secondary antibody, the background signal is unacceptably high. Thus despite the
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Huff Sheela
National Blood Authority
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