Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-13
2001-06-26
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007940, C435S018000, C435S021000, C435S962000
Reexamination Certificate
active
06251618
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of color development using the reaction of an indolyl derivative as a color developing substrate with an enzyme in the presence of a free radical compound and/or a chelate compound, an enzyme immunoassay (hereinafter abbreviated as EIA) utilizing the method of color development, and an immunochromatography incorporating the enzyme immunoassay.
BACKGROUND OF THE INVENTION
An indolyl derivative has been used as a color developing substrate in immunoblotting or immunochromatography (see Methods in Enzymology, Vol. 121, pp. 497-509 (1986) and JP-A-9-133681 which corresponds to EP-A-0762123, the term “JP-A” as used herein means an “unexamined published Japanese patent application”). In these techniques, the activity of an enzyme immobilized on a nitrocellulose membrane through immune reaction is determined by the use of an indolyl derivative. The indolyl derivative reacts with the immobilized enzyme to form an indigoid dye, which is deposited on the membrane with the passage of time to visualize the reaction product.
Immunoblotting requires 20 to 60 minutes from the start of the reaction between an indolyl derivative and an enzyme to color detection, and it has been desired to shorten the reaction time. In immunochromatography, it takes at least 10 minutes, usually from 15 minutes to several hours, for dot blotting a membrane with a sample, development with a developing solution containing an indolyl derivative, and measuring the color developed at the detecting portion to obtain a result. In order to take full advantage of the simplicity characteristic of immunochromatography in urgent examination, it has also been desired to further shorten the time of assaying.
SUMMARY OF THE INVENTION
A first object of the present invention is to provide an improved color developing method using the reaction of an indolyl derivative with an enzyme in the presence of a free radical compound and/or a chelate compound.
A second object of the present invention is to provide an enzyme immunoassay utilizing the above color developing method.
A third object of the present invention is to provide an immunochromatography incorporating the above enzyme immunoassay.
As a result of extensive studies to achieve the above-described objects, the present inventors have found that the time required for color development can be reduced by the reaction of an indolyl derivative with an enzyme in the presence of a specific free radical compound and/or a specific chelate compound.
That is, the present invention provides a color developing method comprising reacting an indolyl derivative represented by formula (I):
wherein R
1
represents an enzymatically releasable group; and R
2
and R
3
each represents a hydrogen atom or a halogen atom,
and an enzyme in the presence of a free radical compound represented by formula (II):
wherein A represents a substituted or unsubstituted methylene chain having 2 or 3 carbon atoms which may contain in its chain an oxygen atom or a carbonyl group; and R
4
, R
5
, R
6
, and R
7
each represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,
and/or a chelate compound formed between a diamine derivative represented by formula (III):
wherein R
8
represents an ethylene group or a cyclohexane-1,2-diyl group,
and a metal selected from the group consisting of iron, copper, zinc, cobalt, indium, neodymium, manganese and europium.
The present invention also provides an enzyme immunoassay comprising measuring enzyme activity of an enzyme-labelled antibody or antigen by using the reaction of the indolyl derivative represented by formula (I) with an enzyme, wherein said reaction is carried out in the presence of the free radical compound represented by formula (II) and/or the chelate compound formed between the diamine derivative represented by formula (III) and a metal selected from the group consisting of iron, copper, zinc, cobalt, indium, neodymium, manganese and europium.
The present invention further provides an immunochromatography comprising measuring enzyme activity of an enzyme-labelled antibody or antigen which is bound to a detecting portion on a solid phase by using the reaction of the indolyl derivative represented by formula (I) with an enzyme, wherein said reaction is carried out in the presence of the free radical compound represented by formula (II) and/or the chelate compound formed between the diamine derivative represented by formula (III) and a metal selected from the group consisting of iron, copper, zinc, cobalt, indium, neodymium, manganese and europium.
REFERENCES:
patent: 5089391 (1992-02-01), Buechler et al.
patent: 5370994 (1994-12-01), Stewart et al.
patent: 5683988 (1997-11-01), Chung
patent: 5807993 (1998-09-01), French et al.
Ashihara Yoshihiro
Miyazaki Mamoru
Sugiyama Masami
Ceperley Mary E.
Fujirebio Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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