Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1987-03-26
1991-08-27
Saunders, David A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
427 2, 435 792, 435973, 435 72, 435 721, 436501, 436524, 436547, 436800, 436805, 530389, 530391, 536 27, G01N 33533, G01N 33551
Patent
active
050432656
DESCRIPTION:
BRIEF SUMMARY
The invention relates to macromolecules, such as proteins, including immunologically specific antibodies, lipoproteins, polynucleotides, etc., provided with luminescent labels.
The invention further relates to a process for preparing macromolecules, such as proteins, including immunologically specific antibodies, lipoproteins, polynucleotides, etc., provided with luminescent labels, and also to the use of labelled immunologically specific macromolecules, such as antibodies, for immunological or immunocytochemical assays.
In biomedical examinations, use is often made of the specific immunological reaction between antigen and (monoclonal) antibody. To render visible and quantify this reaction, the antigen or the antibody is provided with a label. The most common labels are enzymes that can be demonstrated with specific colour reactions, luminescent compounds, and radioactive isotopes. Prominent among luminescent labels are photoluminescent compounds, such as fluorochromes, although the suitability of bioluminescents and chemiluminescents has been demonstrated (Pratt et al., 1978; Simpson et al., 1979; Hastings and Wilson, 1976).
Apparatus for the qualitative and/or quantitative processing of fluorescence signals (photons) has been greatly improved in the last few years. Single-photon-counting detection systems and highly sensitive cameras of the SIT or ISIT type (with image intensifiers), as well as high-energetic excitation sources, such as lasers, are at present available. To increase the amount of fluorescence, new fluorochromes, such as phycobiliproteins, with a high fluorescence efficiency have been developed (Oi et al., 1983). Polystyrene or latex micropheres, which contain thousands of fluorochrome molecules, have also been introduced as cell labels (so-called Covaspheres) (Molday et al., 1975; European Patent Application EP 0002963 (Eastman Kodak Company), 1979). Thanks to these improvements, the extreme sensitivity of fluorescence studies of cells and tissues is at persent determined for the major part by the autofluorescence of the biological object and the optics used (lenses and filters), and by undesirable excitation light from the radiation source. The autofluorescence of a single cell, measured with a microscope fluorimeter or a flow cytometer, is in the order of several thousands of fluorescein equivalents (Jongkind et al., 1979), as a result of which the demonstration of minute amounts of macromolecule in cells and tissues by means of immunofluorescence is not optimally possible. It is especially for the demonstration of minute amounts of macromolecules, therefore that one would often opt for a method using a radioactive label. This method is sensitive, it is true, but in the case of in situ applications (e.g. autoradiography) extremely time-consuming, while in addition the necessary precautionary measures must be taken with regard to working conditions and the processing of the radioactive waste.
In analytical chemistry, the problem of autofluorescence can be largely avoided by using so-called time-resolved fluorescence assays. For this method a luminescent label must be selected with a long after-glow time, such as the lanthanides europium (eu) and terbium (Tb). After a short exciting light pulse, the relatively fast autofluorescence (in the order of nanoseconds or faster) can be separated in time from the slower lanthanide fluorescence (in the order of milliseconds). The DELFIA (Delayed Fluoro Immuno Assay) system, developed by LKB, makes use of this principle. This method has been found to be competitive in sensitivity with radioactive assays (Soini et al., 1983; Hemmila et al., 1984). One disadvantage is, however, that the luminescent label Eu or Tb when bonded to antibodies and dissolved in water does not luminesce, or hardly so, and therefore after the reaction with the antigen-antibody must be liberated to obtain the delayed-fluorescence properties. As a consequence, detection is only possible in solutions, and uses of this method in which localisation of the antigen at the tissue, c
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Ploem Johan S.
Slats Johannes C.
Tanke Hendrikus J.
501 Rijksuniversiteit leiden
Saunders David A.
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