Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-18
2002-11-19
Borin, Michael (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S512000, C436S546000, C436S172000, C435S007500, C435S007900, C435S007920, C435S007940
Reexamination Certificate
active
06482596
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring antigen concentration in a sample. It relates more specifically to a new method for making more simple and highly sensitive measurements of antigen concentration by employing intramolecular interactions between components of antibody (immunoglobulin). This method can be utilized as, for example, a sandwich ELISA method, and is useful for clinical diagnosis of the concentrations of various antigens in biological samples (blood, urine and body fluid). It is also applicable to the specific detection of all other sorts of substances, both natural and unnatural, recognizable as an antigen, and can be used as research and industrial sensors.
DESCRIPTION OF RELATED ART
Conventionally, various methods for measuring antigen concentration have been known, some of which are used for clinical diagnosis. Of these methods for measuring antigen concentration, the one commonly called sandwich ELISA method (or sandwich PTA method) attracts special attention. This method is characterized by measuring the concentration of antigen using two kinds of monoclonal antibodies which recognize different epitopes of the antigen, alternately, with one kind of monoclonal antibody and one kind of polyclonal antibody. The procedure of this sandwich ELISA consists of three stages. In the first stage, an antigen-containing sample is poured on a measurement plate on which monoclonal/polyclonal antibodies (primary antibodies) have been adsorbed; the antigens in sample are bound to the primary antibodies. In the second stage, the substances in the sample other than the antigen are washed off with a washing agent. Then, in the third stage, a solution of the secondary antibodies, labeled with reporter molecules, such as an enzyme or radioisotope, are poured on the plate; the labeled antibodies bind to the antigens having been bound to the primary antibodies.
Excessive labeled antibodies are fully rinsed away with washing agent, then the amount of the reporter molecules left in the measurement plat 0 is measured by means of an enzyme activity reader or a liquid scintillation counter; and the observed values are used for the estimation of the quantity of antigens in the sample.
The conventional sandwich ELISA method consists of the operation steps as described above, and it has the advantage of being capable of measuring essentially all kinds of protein antigens, so long as the necessary antibodies are available. On the other tend, however, the measurement procedure Involves at least two cycles of reaction and washing, and inevitably becomes complicated, time-consuming and costly when it is to be automated.
SUMMARY OF THE INVENTION
In order to solve the aforestated problems associated with the conventional ELISA method, the present invention provides a new method which permits simpler and speedier measurements of antigen concentration.
The first method according to the instant invention is a method for measuring an antigen concentration in a sample, which comprises:
(a) preparing VH-domain polypeptide and VL-domain polypeptide of an antibody specific to the antigen;
(b) labeling one of the polypeptides with a reporter molecule to form labeled polypeptide, and immobilizing the other polypeptide onto a solid-phase to form immobilized polypeptide;
(c) contacting the antigen-containing sample and the labeled polypeptide with the solid-phase; and,
(d) measuring the reporter molecule of the labeled polypeptide bound to the immobilized polypeptide.
One embodiment of the first method in this invention is that the polypeptide to be immobilized onto solid-phase is preferably biotinylated or modified with a tag sequence, and the solid-phase adsorbs or chemically bonds avidin or streptavidin, thereby immobilizing the polypeptide onto the solid-phase through biotin-avidin binding or tag sequence-streptavidin binding.
Another embodiment of the first method is that the reporter molecule is filamentous phage which is fused to the polypeptide, in which case the filamentous phage is preferably labeled with an enzyme or a fluorochrome.
A further embodiment of the first method is that the reporter molecule is an enzyme, in which case the enzyme is preferably alkaline phosphatase originated from
Escherichia coli.
A further embodiment of the first method is that the reporter molecule is a fluorochrome, in which case the fluorochrome is fluorescein or a derivative thereof.
The second method according to the present invention is a method for measuring an antigen concentration in a sample, which comprises:
(a) preparing VH-domain polypeptide and VL-domain polypeptide of an antibody specific to the antigen;
(b) labeling tho VH-domain polypeptide with a 1st reporter molecule, and labeling the VL-domain polypeptide with a 2nd reporter molecule;
(c) mixing the labeled VH-domain polypeptide and the labeled VL-domain polypeptide with the antigen-containing sample; and
(d) measuring a changed characteristic value of the 1st reporter molecule or the 2nd reporter molecule which is induced by the interaction of both molecules.
One embodiment of the second method in this invention is that the 1st reporter molecule and the 2nd reporter molecule are fluorochromes of different kinds, and change of fluorescence intensity induced by energy transfer between both fluorochromes is used to measure the amount of the VH-VL complex. In this case, the fluorochromes are preferably fluorescein or a derivative thereof and Rhodamine or a derivative thereof.
Having the aforestated constituents, the first and the second methods according to the present invention describe a new ELISA process which permits simpler and quicker measurements of antigen concentration while offering the same or higher level of sensitivity and reliability as compared with the sandwich ELISA method conventionally available. The present invention was based on the phenomenon, as previously unnoticed, that the stability of the variable Fv-domain regions (VH-domain region and VL-domain region) of an antibody varies significantly depending upon the presence or absence of an antigen to be bound to the Fv-domain, and this invention makes active use of this property to conduct measurements of the concentration of antigen.
In addition, the present invention also provides a labeled polypeptide, a set of labeled polypeptides and a kit for measuring an antigen concentration in a sample.
Then, the present invention will be described in detail, in which VH-domain polypeptide may be referred to as VH, and VL-domain polypeptide as VL.
REFERENCES:
patent: 4859609 (1989-08-01), Dull et al.
patent: 5030576 (1991-07-01), Dull et al.
patent: 5196510 (1993-03-01), Rodwell et al.
patent: 5424192 (1995-06-01), Thompson et al.
patent: 5639660 (1997-06-01), Kinet et al.
patent: 5948894 (1999-09-01), Berry et al.
patent: 5-184386 (1993-07-01), None
Database Caplus, DN 122:26912; Szollosi, J. et al. Cytometry (1998), 34(4), 159-179, Apr. 1998.*
Database Caplus, DN 129:327783; Tron L. Mobility Proximity Biol. Membr. (Jan. 1994), 1-47 Publisher: CRC, Boca Raton, Fla.*
Database Caplus, DN : 121:106236; Mallender et al. Biochemistry (1994), 33(33), 10100-8, Feb. 1994.*
Anthony, J. et al., (1992) “Production of stable anti-digoxin Fv inEscherichia Coli” Mol. Immunol.29(10):1237-1247.
Better, M. et al., (May 20, 1988) “Escherichia colisecretion of an active chimeric antibody fragment”Science240:1041-1043.
Bird, R.E. et al., (Oct. 21, 1988) “Single-chain antigen binding proteins”Science242:423-426.
Brinkmann, B. K. et al., “Recombinant immunotoxins containing the VHor VLdomain of monoclonal antibody B3 fused to Pseudomonas exotoxin”J. Immunol. 150(7):2774-2782 (1993).
Chothia, C. et al., (1985) “Domain association in immunoglobulin molecules. The packing of variable domains”J. Mol. Biol.186(3):651-663.
D'Andrea, A.D. et al. (1989) “Expression cloning of the murine erythropoietin receptor”Cell57:277-285.
Davies, J. et al., (1994) “‘Camelising’ human antibody fragments: NMR studies on VH domains”FEBS Letters339:285-290.
Figini, M. et al., (
Kumagai Izumi
Mahoney Walter C.
Nagamune Teruyuki
Nishimura Hajime
Schueler Paula A.
Borin Michael
Roche Diagnostics Corporation
Wenderoth , Lind & Ponack, L.L.P.
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