Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-01-09
1992-05-19
Rosen, Sam
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, C12Q 128
Patent
active
051148412
DESCRIPTION:
BRIEF SUMMARY
This invention relates to improvements in luminescent or luminometric bioaffinity assays, for example immunoassays, and to diagnostic kits whereby such assays can be readily performed.
An immunoassay enables the detection of an analyte which may be an antibody or an antigen. An antigen may be detected by using an antibody which is specific to that antigen. Alternatively, immunoassays can be used to determine antibodies by utilizing their specific binding to a particular antigen. At first, either antibodies or antigens were covalently linked to radioactive atoms which acted as labels enabling quantitative detection of the labelled component in the resulting antigen/antibody complex. However, radioactive detection systems suffer from a number of important disadvantages. For example, the radioactive agents required are inherently hazardous and can present problems of disposal. Further, as the radioisotopes normally used have a relatively-short half life, they can be stored for only limited periods. These isotopes are also expensive.
Partly as a result of these and other disadvantages of using radioactivity as a label in immunoassay systems, certain alternative detection systems have been developed. Some of these systems utilize as labels compounds that can take part in chemical reactions which result in the emission of light. The light emitted by the reaction can be quantitatively measured to give an accurate estimate of the amount of labelled immunoreactant bound which reflects the amount of analyte present in the sample, enabling it to be quantitatively measured by the assay.
One type of luminescent immunoassay is based on the reaction between a per compound as oxidant (e.g. hydrogen peroxide), a peroxidase catalyst, and a luminescent 2,3-dihydro-1,4 phthalazinedione (herein abbreviated to DPD), in which the peroxidase can act as the label.
Various modifications and improvements of the above-mentioned luminescent system have been proposed. For example, GB-A-2162946 (National Research Development Corporation), EP-A-116454 and EP-A-87959 (Secretary of State for Social Services) disclose the use of certain specifically-defined aromatic compounds as so-called "enhancers" of the chemiluminescence resulting from the above reaction. One of the enhancers mentioned in GB-A-2162946 is 3,3',5,5'-tetramethylbenzedine (commonly referred to as TMBZ). Another is N,N,N',N'-tetramethylbenzidine (NNNN-TMBZ). In the reactions described in the above patent applications light emission rises rapidly to a maximum intensity value, which persists for a considerable length of time as a constant or "plateau" value, for example for several minutes. One possible advantage of constant emission of light over an extended period is that the chemical reaction can be carried out outside the luminometric apparatus and then transferred to that apparatus for measurement when emission of light is already taking place. On the other hand, extended light emission can be disadvantageous in certain circumstances. Thus, if a series of test reactions is being carried out in the wells of a conventional microtiter plate, and if as a result of extended photoluminescence several of the wells contain glowing reaction mixture at the same time, there is a danger of "cross-talk" between two or more wells. In other words, there is a danger of stray light from one or more adjacent wells interfering with the measurement of the luminescence that is being carried out on the contents of a particular well.
It is to be noted that in the assay procedure described in the above patents, the reaction between the peroxidase, the oxidant, the chemiluminescent DPD and the specific aromatic enhancer is carried out in a single step.
We have now discovered that if an aromatic substrate, which may be one of the "enhancers" that can be used in the assay procedures disclosed in the above-mentioned patent applications, the peroxidase and the per compound are reacted together in a first step, an intermediate product is obtained, and that subsequent addition of the DPD at an appropri
REFERENCES:
patent: 4598044 (1986-07-01), Kricka et al.
patent: 4729950 (1988-03-01), Kricka et al.
Blomberg Fred
Friberg Jan
Glindre Jan-Olof
Kangasmetsa Jarl
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