Method of preferential labelling of a phycobiliprotein with an amine-reactive dye for use in a multiple color assay and product for such use

• US 5,272,257 A
• Filed: 09/03/1992
• Issued: 12/21/1993
• Est. Priority Date: 05/21/1990
• Status: Expired due to Term

• Current Assignee: Coulter International Corp. (Danaher Corporation)
• Original Assignee: Coulter Corporation (Danaher Corporation)
• Inventors: Gupta, Ravinder K.
• Primary Examiner(s): KIM, KAY KWANG AE
• Application Number:
  US07/940,026
• Time in Patent Office: 474 Days
• Field of Search: 530/403, 530/409, 530/410, 530/370, 530/400, 530/401, 530/395, 530/404, 530/405, 530/408, 530/345, 530/402, 436/546, 436/501, 436/536, 436/543, 436/544, 436/545, 435/961, 435/964
• US Class Current: 530/370
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein

Abstract

A method for preparing phycobiliprotein/amine-reactive dye conjugates is disclosed in which the conjugates so prepared overcome the energy transfer/fluorescent quenching dilemma encountered in the use of prior art conjugates. A phycobiliprotein, for example, phycoerythrin or allophycocyanin, is conjugated with an amine-reactive dye, for example, Texas Red or carboxyfluorescein succinimidyl ester, in the presence of a selective salt which causes a hydrophobic intramolecular rearrangement of the phycobiliprotein thereby exposing more hydrophobic sites for binding to the amine-reactive dye. The conjugates prepared according to the invention are useful in multiple color fluorescence assays without requiring the use of multiple exciting sources.

Claims

1. A method for producing a phycobiliprotein/amine-reactive dye conjugate wherein said phycobiliprotein is labelled with said dye, said method comprising reacting an amine-reactive dye with a phycobiliprotein in the presence of 1 to 20% of salt which effects an intramolecular rearrangement of said phycobiliprotein to expose reactive amino groups in hydrophobic sites on said phycobiliprotein which reacts with said dye, removing excess dye and separating the conjugate by a chromatographic means.

2. The method of claim 1 wherein a molar ratio of said dye to said phycobiliprotein att he start of said reaction is in the range of 5:

1 to 30;

1.

3. The method in accordance with claim 1 wherein the salt is one whose anion is selected from the group consisting of phosphate, sulfate, acetate, citrate and tartrate ions.

4. The method of claim 1 wherein said salt has a concentration in the range of 1% to 12%.

5. The method of claim 1 wherein said chromatography is hydrophobic interaction chromatography.

6. The phycobiliprotein/amine-reactive dye conjugate produced according to claim 1.

7. A method for producing a phycobiliprotein/amine-reactive dye conjugate suitable for use in a multiple color assay wherein said phycobiliprotein is labelled with said dye, said method comprising reacting a phycobiliprotein containing solution of pH greater than 7 and a molar excess of an amine-reactive dye in the presence of 1% to 20% of a salt at a temperature in the range of 4°

C. to 25°

C., removing unreacted dye and separating the conjugate by a chromatographic means.

8. The method in accordance with claim 7 wherein said salt is a salt whose anion is selected from the group consisting of phosphate, acetate, sulfate, citrate, and tartrate.

9. The method of claim 7 wherein said salt concentration is in the range of 1% to 12%.

10. The phycobiliprotein/amine-reactive dye conjugate produced according to claim 7.

11. A method for producing a phycobiliprotein/amine-reactive dye conjugate wherein said phycobiliprotein is labelled with said dye, said method comprising reacting a molar excess of an amine-reactive dye with a phycobiliprotein having reactive amino groups in hydrophobic sites exposed by a salt-induced molecular rearrangement in the presence of 1 to 20% of the salt, said reaction occurring at a pH of greater than 7, removing unreacted dye and separating the conjugate by hydrophobic interaction chromatography.

12. The method of claim 11 wherein the molar ratio of said dye to said phycobiliprotein at the start of said reaction is in the range of 5:

1 to 30;

1.

13. The phycobiliprotein/amine-reactive dye conjugate produced according to claim 11.