Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-08-21
2000-06-27
Hutzell, Paula K.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 435 72, 435 771, 435 794, 435 795, G01N 3353
Patent
active
060805528
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the detection of antibody production, and in particular to the detection of active antibody synthesis in blood samples in response to infection or vaccination etc., by means of a modified enzyme-linked immunosorbent assay (ELISA).
BACKGROUND ART
ELISA has long been used to detect and measure antibody (or antigen). Most commonly, ELISA is used as a serological assay, but it is also used to study the immunochemical properties of antigens or antibodies, and has frequently found application in, for example, the evaluation and characterisation of immune responses, to investigate antibody production by cell cultures, in hybridoma technology etc.
Due to its sensitivity, simplicity and ease and speed of operation, the technique has been widely adopted as a diagnostic tool and is now routinely used in clinical laboratories to detect antibodies in serum or plasma to infectious agents, following infection or vaccination. Thus for example, many tests for HIV infection depend upon detecting antibodies to the virus in the serum or plasma of patients using a conventional ELISA assay.
However, since such a simple serological ELISA test simply measures the presence of the target antibody in the sample, it cannot distinguish between on-going antibody synthesis in response to the antigen, and antibodies already present from past infection, or by passive transfer etc. Whilst in some cases, it may suffice simply to obtain information concerning the presence of antibody, it is in other cases desirable to be able to determine whether or not the detected target antibodies are acutely synthesised by the lymphocytes at the time of testing, for example during a vaccination course, or in the diagnosis of infection in infants, to distinguish from passively transferred maternal antibodies. This cannot be achieved in a classical ELISA method.
Other methods have therefore been developed, which enable on-going antibody synthesis to be detected. Particular mention may be made in this regard of the enzyme-linked immunospot (ELISPOT) assay (also known as spot ELISA or ELISA-plaque assay), as reviewed for example by Czerkinsky et al. in ELISA and other Solid Phase Immunoassays, Ed. D. M. Kenneny and S. J. Challacombe, 1988, Chapter 10, 217-239. This technique, based on the ELISA method, enables the enumeration of lymphocytes secreting antibody against one or more target antigens. Basically, the ELISPOT is a variant of the ELISA method, whereby antibody secreting cells (ASC) may be revealed by culturing lymphocytes in specially modified ELISA wells coated with the target antigen, and by replacing the standard ELISA reagents with enzyme-substrate complexes that yield a coloured precipitate (spots), adjacent to the secreting cell. Spots can then be counted to give a measure of the number of antibody-producing cells. Protein synthesis inhibitors may be included in the culture medium, to confirm that the spots detected are due to de novo antibody synthesis, during the in vitro incubation period.
Whilst the ELISPOT technique has proved very useful in studying the dynamics of humoral immune responses, and has been used to detect spontaneous ASC that appear transiently in the peripheral circulation of immunised subjects, certain features of the method place constraints on its use in a clinical diagnostic setting. Firstly, since for each sample individual spots need to be counted which can be time consuming and laborious, the method is not particularly suited to the analysis of large numbers of samples, such as occurs in a clinical diagnostic laboratory. Secondly, only the number of cells secreting antibody in each sample is enumerated and generally speaking, this requires reasonably large sample volumes, eg. several mls. ELISPOT plates are also expensive and the assay is not readily amenable to automation.
It will be seen therefore, that despite advances in antibody detection techniques, there remains a need for an assay which is simple, quick and cost effective to perform, which reliably enables p
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Hutzell Paula K.
Sun-Hoffman Lin
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