US4463099A
Expired / Lapsed
US
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Immunofluorescence reagents, and the method for their preparation
Assignee: Sclavo SpA
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Filing: 1982-09-16
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Granted: 1984-07-31
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Expires: 2002-09-16
Abstract
Abstract New immunofluorescence reagents prepared by reacting together a protein, a cross-linking agent and a dye, which are of general formula ##STR1## and which fluoresce between 600 and 900 nm.
Inventors
- 👤 Vittorio Baroncelli
- 👤 Claudio Colapicchioni
- 👤 Ivo Giannini
- 👤 Filippo Porcelli
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 15:29
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
209 words
Immunofluorescence reagents of general formula ##STR10## in which n is a whole number between 1 and 4 n is a whole number equal to n or o P is a protein L is the residue of a cross-linking agent X is an oxygen or sulphur atom Y is a group chosen from NH, NH 2 , NR' and NR'R", where R' and R", which can be the same or different, are substituted and unsubstituted alkyl, aryl, arylalkyl or alkylaryl radicals, or a OCOR' group where R' has the aforesaid meaning; R 1 , R 2 , R 3 , R 4 , R 5 , R 7 and R 8 , which can be the same or different, are either a hydrogen atom or an amino group or a substituted or unsubstituted alkyl, aryl, alkylaryl or arylalkyl radical, or R 1 and R 2 and/or R 7 and R 8 are bivalent radicals which close together to thus form a condensation ring with the cycloaromatic ring; at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 7 and R 8 must be an amino group which on reacting with the cross-linking reagent gives rise to --NH--L or N═L group.
Claim 2
Dependent Claim
209 words
Immunofluorescence reagents of general formula ##STR10## in which n is a whole number between 1 and 4 n is a whole number equal to n or o P is a protein L is the residue of a cross-linking agent X is an oxygen or sulphur atom Y is a group chosen from NH, NH 2 , NR' and NR'R", where R' and R", which can be the same or different, are substituted and unsubstituted alkyl, aryl, arylalkyl or alkylaryl radicals, or a OCOR' group where R' has the aforesaid meaning; R 1 , R 2 , R 3 , R 4 , R 5 , R 7 and R 8 , which can be the same or different, are either a hydrogen atom or an amino group or a substituted or unsubstituted alkyl, aryl, alkylaryl or arylalkyl radical, or R 1 and R 2 and/or R 7 and R 8 are bivalent radicals which close together to thus form a condensation ring with the cycloaromatic ring; at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 7 and R 8 must be an amino group which on reacting with the cross-linking reagent gives rise to --NH--L or N═L group.
Claim 3
Dependent Claim
98 words
A method for preparing the reagents of claim 1, characterised by reacting together at least one compound chosen from each of the following classes: (a) a protein; (b) a dye chosen from those oxazines or thiazines which in the visible range have an absorption with λ max between 550 and 800 nm, and which have at least one free NH 2 , and a fluorescence with emission between 600 and 900 nm (c) a cross-linking agent, generally in the form of bifunctional reagents of which the groups are able to react with the amino group of the dye.
Claim 4
Dependent Claim
40 words
A method for preparing reagents as claimed in claim 2, characterised in that the reaction between the three compounds can be carried out either starting from the substrates alone or in the presence of a medium compatible with the compounds.
Claim 5
Dependent Claim
37 words
A method for preparing reagents as claimed in claim 3, characterised in that the reaction is preferably carried out in the presence of water, or of aqueous solution of one or more salts, or of buffer mixture.
Claim 6
Dependent Claim
26 words
A method for preparing reagents as claimed in claim 3, characterised in that the reaction takes place at a pH which varies from 4.5 to 9.5.
Claim 7
Dependent Claim
20 words
A method for preparing reagents as claimed in claim 5, characterised in that the reaction temperature varies around ambient temperature.
Claim 8
Dependent Claim
26 words
A method for preparing reagents as claimed in claim 6, characterised in that the reaction temperature varies preferably between 4° C. and the protein denaturation temperature.
Claim 9
Dependent Claim
27 words
A method for preparing reagents as claimed in claim 7, characterised in that the reaction is carried out with a large excess of dye over the protein.
Claim 10
Dependent Claim
27 words
A method for preparing reagents as claimed in claim 8, characterised in that the reaction is varying from 4 to 50 times in terms of molar concentration.
Claim 11
Dependent Claim
36 words
A method for preparing reagents as claimed in claim 9, characterised in that the reaction is carried out with a large excess of the bifunctional agent over the total quantity of protein and dye taken together.
Claim 12
Dependent Claim
17 words
A method for preparing reagents as claimed in claim 1 wherein R 3 is an amino group.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1987-07-31 | 1988-01-30 | 1988-07-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1991-07-31 | 1992-01-30 | 1992-07-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1995-07-31 | 1996-01-30 | 1996-07-31 | $7700.00 | Window Closed / Lapsed |